BioXp 3200 System - Codex DNA · Provide means to promptly and safely dispose of any flammable liquid leaks or spills. Do not transfer liquids using air pressure. Read applicable

BioXp™ 3200 SystemUser's Guide

REV. 5.0 01.08.19Part number 40010

LEGAL NOTICES

Limited Use Label License Except as otherwise agreed in writing by our authorized representative, this product is for INTERNAL RESEARCH USE ONLY AND NOT FOR HUMAN, ANIMAL, THERAPEUTIC OR DIAGNOSTIC USE. For additional information about your rights under this research license, please see our website at sgidna.com.

Limited Warranty a) SGI-DNA's entire liability and your exclusive remedy if the Product fails to conform to the following warranty (a “Nonconforming Product”) shall be, at SGI-DNA's sole option, either repair or replacement of such Nonconforming Product, or, if neither is practicable, a refund of the fees paid for the Product. The warranty for the repaired or replaced Product is limited to the scope and remaining duration of the original warranty for the Nonconforming product or, if longer, for 30 days after the date of shipment to you of the repaired or replaced Product. The warranty is contingent upon proper use of the Product in accordance with the User's Guide and does not apply to any Product that is subjected to unusual physical or electrical stress, misuse, neglect, improper testing or storage, modification or unauthorized repair or upgrade.b) SGI-DNA warrants that the product, as delivered, will be free from defects in material and workmanship for a period of one year from the date it is delivered to you (“the Warranty Period”).c) This warranty gives you specific legal rights and you may have other legal rights which vary from state to state. This warranty is non-transferable.d) OTHER THAN AS EXPRESSLY SET FORTH ABOVE, SGI-DNA MAKES NO WARRANTIES, EXPRESS, STATUTORY, IMPLIED, OR OTHERWISE. NO DISTRIBUTOR, AGENT OR EMPLOYEE IS AUTHORIZED TO MAKE ANY MODIFICATIONS, EXTENSIONS OR ADDITIONS TO THIS WARRANTY.e) You shall indemnify, defend and hold harmless SGI-DNA from any costs, expenses, damages, or other losses arising out of i) any warranty of greater scope or duration than that set forth in this SGI-DNA Limited Warranty; and ii) failure to disclaim implied warranties and limit remedies and liabilities, by and on behalf of SGI-DNA.

Trademark Information TipOne® is a registered trademark of USA Scientific, Inc.Gibson Assembly® is a registered trademark and BioXp™, Oligo Vault™, and Bio360™ are trademarks of SGI-DNA.E. cloni® is a registered trademark of Lucigen Corporation.TransforMax™ and EPI300™ are trademarks of Epicentre, Madison, Wisconsin.Gene Pulser® is a registered trademark and Xcell™ is a trademark of Bio-Rad Laboratories, Inc.Qubit® is a registered trademark and Nunc™ is a trademark of Thermo Fisher Scientific Inc.Q5® and OneTaq® are registered trademarks of New England BioLabs, Inc.

Regulatory Statement For Research Use Only

Statement of Proprietary Information

Copyright in this work is vested in SGI-DNA and the document is issued in confidence for the purpose only for which it is supplied. It must not be reproduced in whole or in part except under an agreement or with the consent in writing of SGI-DNA and then only on the condition that this notice is included in any such reproduction. No information as to the contents or subject matter of this document or any part thereof arising directly or indirectly there from shall be given orally or in writing or communicated in any manner whatsoever to any third party being an individual firm or company or any employee thereof without the prior consent in writing of SGI-DNA.

Terms and Conditions IMPORTANT- This agreement (the “Agreement”) is a legal agreement between you and SGI-DNA. By installing or otherwise using the BioXp™ 3200 System (referred in this agreement as the “Product”), you agree to be bound by the terms of this Agreement. If you do not wish to be bound by the terms of this Agreement, do not use the Product and promptly return the unused, unopened Product to the place of purchase for a full refund. The user is prohibited from reverse engineering the software or output files generated from the BioXp™ 3200 system.

Limitation of Liability SGI-DNA'S LIABILITY ARISING OUT OF OR RELATING TO A PRODUCT SHALL NOT EXCEED THE AGGREGATE AMOUNTS YOU PAID TO SGI-DNA FOR THE PRODUCT. IN NO EVENT WILL SGI-DNA BE LIABLE FOR LOST USE, PROFITS, REVENUE, COST OF PROCUREMENT OF SUBSTITUTE GOODS, OR ANY OTHER SPECIAL, INDIRECT, RELIANCE, INCIDENTAL, OR CONSEQUENTIAL DAMAGES, HOWEVER CAUSED AND UNDER ANY THEORY OF LIABILITY. THE FOREGOING LIMITATIONS SHALL APPLY REGARDLESS OF WHETHER SGI-DNA HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AND NOT WITHSTANDING THE FAILURE OF ESSENTIAL PURPOSE OF ANY LIMITED REMEDY.

Disclaimer The material in this manual is for informational purposes only and is subject to change without prior notice at any time. SGI-DNA and/or its affiliates assume no responsibility for any errors that may appear in this document.

INDICATIONS FOR USE The BioXp™ 3200 System is a device which is intended for the processing of DNA materials. The system is not approved for use other than that stated above.

Technical Services For technical assistance, please contact technical services at [email protected] or 1-855-474-4362.

SGI-DNA sgidna.com

©2019 SGI-DNA. All rights reserved.Gibson Assembly®: US Patent Nos. 7,776,532, 8,435,736, and 8,968,999.

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Table of Contents

BioXp™ 3200 Instrument Guide 3

Instrument Ordering Information 3

Safety Information 3Manual Symbols 3Precautions 3Marking Symbols on the Equipment 3Safety Warnings 4

Equipment Specifications 6System Components 6Technical Specifications 6

Installation Guide 6System Location 6Unpacking, Set up, and Power Up 8Getting to Know the System 9

Starting the BioXp™ System 10Ethernet Set Up 10Start the BioXp™ System 10

Quick Start Guide: Performing an Instrument Run 11BioXp™ Tiles and Libraries Quick Start 11BioXp™ Cloning Quick Start 12

Operational Features of the BioXp™ System 15Delay Start 15Tracking Progress During a Run 16Canceling a Job During Delay Start 17Canceling a Job During a Run 17Shutting down the BioXp™ System 17

Configuring the BioXp™ System 18

Instrument Troubleshooting 19General Instrument Troubleshooting 19Deck Inspection 19Error Messages 19

Cleaning and Maintenance 20Cleaning Instructions 20Calibration 20Preventative Maintenance 20Repair 20Recommended Separation Distances 20

Equipment Disposal 21WEEE Compliance 21

Regulatory Statements 22FCC Compliance Statement 22Guidance and Manufacturer’s Declaration: Emissions 22Guidance and Manufacturer’s Declaration: Immunity 22

1

BioXp™ Tiles, Libraries, and Clones 23

Product Ordering Information 23

Overview 23Receiving an Order 23Required materials (not included in the product order) 24Specifications 24Consumables and Reagents 25

Instrument Operation 26Preparing for a BioXp™ 3200 System run 26Load the instrument 27Begin the Run 28During the Run 28After the Run 28

Analyzing BioXp™ Tiles, Libraries, and Clones 30Gel Electrophoresis 30

Next Steps– BioXp™ Tiles and Libraries 31Quantification 31Cloning BioXp™ Tiles 31Multi-fragment Assembly 31PCR Amplification (Optional) 32Colony Screening (Optional) 33

Next Steps– BioXp™ Clones 34Transformation 34

Full-length Clone (EFC) Analysis 36Screening colonies 36

Reference Material 38pUCGA 1.0 Amp clone map 38

Troubleshooting 39

2

3

BioXp™ 3200 Instrument Guide

Instrument Ordering InformationProduct Description Cat. No.

BioXp™ 3200 System, includes 1 year warranty Standard Ethernet configuration of BioXp™ Instrument

BX 3200-02 andBX 3200-02 EU

BioXp™ 3200 System Service Contract, 1 year Covers parts and labor for instrument failure for 1 year, renewable annually

BX 3200-05 and BX 3200-05 EU

Safety InformationManual Symbols

This manual describes the use and maintenance of the SGI-DNA BioXp™ 3200 Instrument. Read this manual completely before putting the instrument into service. The following icons are used in this manual:

Electrical Safety Warning: Failure to follow these instructions can cause harm to operators or subjects.

Important Note: Failure to follow these instructions can lead to damage of the system or unexpected results.

Precautions• The BioXp™ System conforms to the following standards: IEC 61010-1:2001 2nd Edition to include

EN 61010-1:2001, IEC/EN 61010-2-010, IEC/EN 61010-2-081.• The radiation exposure hazard is non-critical.

Covers and exterior components which the operator may come in contact with during routine maintenance or calibration, shall operate at a voltage no greater than 25 V AC or 60 V DC.

Marking Symbols on the Equipment

Warning: Indicates a warning concerning operations that may lead to personal injury or potential damage to the instrument if not performed correctly.

Caution, Hot Surface, Do NOT Touch.

Conformité European: This mark is a declaration by the manufacturer that the respective component complies with the relevant directives and standards as issued by the European Union.

Alternating Current.

Waste Electrical and Electronic Equipment (WEEE) Directive: Do not dispose symbol. See "Equipment Disposal" on page 21 for additional information.

Consult instructions for use.

4

Safety WarningsInstallation

The BioXp™ System is heavy. Do not attempt to lift or move the instrument without the assistance and supervision of colleagues. Be certain to use appropriate moving equipment and proper lifting techniques (including but not limited to making certain that you have a secure grip on the instrument before lifting, keeping your spine in a neutral position while lifting with your legs, and coordinating lifting and moving movements with all appropriate personnel). Improper lifting can cause permanent back injury.

Use this product only as specified. If the instrument is used in a manner not specified by SGI-DNA, the protection provided by the instrument may be impaired, resulting in personal injury or damage to the instrument.

Ground Circuit ProtectionCheck the mains electricity supply plug, voltage, and frequency to confirm that the supply corresponds to the values shown on the instrument label.

Ensure that the supply of power to the instrument is via a socket fitted with a protection device, such as a circuit breaker of the correct rating, to provide automatic power cut-off in the cause of an insulation fault.

Confirm that the mains wiring is grounded. SGI-DNA declines all responsibility for any damages due to instrument non-grounding.

Main Power Switch

Do not position the instrument such that it is difficult to operate the main power switch.

Hazardous Chemical WarningChemicals used with the instrument may be hazardous.

Always wear protective gear including safety goggles, gloves, and a lab coat when handling chemicals.

Exercise caution when handling flammable liquids.

Take precautions, including but not limited to installing proper ventilation systems, to ensure that the BioXp™ System laboratory is safe and that operators of the instrument will not be exposed to hazardous levels of harmful chemicals.

Follow all national, state, and local health and safety regulations and laws.

Use proper waste disposal in accordance with all relevant regulations.

Refer to applicable Material Safety Data Sheets (MSDSs).

Flammable Liquids: Instrument WarningExercise caution when using flammable liquids in the BioXp™ System to minimize the risk of fire.

Do not use any flammable liquids in the instrument except for the liquids required in the included procedure.

Provide adequate ventilation for the instrument to prevent the accumulation of flammable vapors.

All sources of ignition, such as open flame or electrical spark, are not permitted near the instrument or in areas where flammable vapors may travel.

Ensure that the instrument, all neighboring equipment, and users of the instrument are appropriately grounded to minimize the generation of static electricity.

Do not use the Ethanol Reservoir if it is cracked.

5

Flammable Liquids: Solution Handling WarningKeep flammable liquids in covered containers when not in use.

Provide means to promptly and safely dispose of any flammable liquid leaks or spills.

Do not transfer liquids using air pressure.

Read applicable MSDSs.

Store flammable liquids in cool, well-ventilated areas away from corrosives, oxidizers, and ignition sources.

Label containers and cabinets as "flammable materials" where applicable.

Use only approved safety vessels for flammable liquid storage.

Ground and bond flammable liquid containers to prevent static charge build-up.

Never pour flammable liquids down a drain or sink.

Dispose of empty flammable containers in an approved manner.

Electrical Safety WarningsDo not attach the power cord to an extension cord or to a multiple portable socket. Doing so may compromise shielding and/or grounding.

This equipment should not be used adjacent to or stacked with other equipment. If it becomes necessary to use the BioXp™ System under these conditions, the unit should be observed to verify normal operation in this condition.

Use only grounded mains outlets for instrument supply.

Use only grounded mains outlets to supply instrument.

Do not remove any panels. Panels should be removed only by trained service personnel.

The AC power cord is the primary disconnection device for the instrument. Ensure that the point of connection is easily accessible and free of any obstructions.

Important Warning: Moving PartsDo not override the door lock. When the door is locked, the moving parts of the instrument are operational. Never attempt to physically restrict movement of instrument components.

Interfering with moving parts may cause personal injury. Keep hands clear of moving parts while operating the instrument.

Do not move or shift the instrument during operation.

If the instrument shows evidence of corrosion or wear, do not attempt to manipulate or tamper with components of the instrument.

Important Warning: Disconnecting the InstrumentDisconnect power to the system before cleaning or performing maintenance.

DO NOT remove any panels; panels should be removed only by qualified service personnel.

Do not disassemble unit.

6

Equipment SpecificationsSystem Components

Use of other parts or materials other than those supplied with the instrument can degrade system performance and may cause safety hazards. Use only SGI-DNA approved or supplied components.

Use only SGI-DNA supplied Power Supply Cord. Use of other power cords may compromise electrical protection and could create a hazard.

Components of the BioXp™ SystemBioXp™ 3200 Instrument

Power Cord

RJ45 Ethernet Cable

Reagent and Recovery Plate Thermal Covers

Ethanol Reservoir

Technical SpecificationsProduct Name BioXp™ 3200 System

Power Input Voltage 100–240 V

Power Input Current 8.3 A Max

Operating Temperature Range 16°C to 40°C

Storage Temperature Range −18°C to 60°C

Operating and Storage Humidity Range 10% to 90% (non-condensing relative humidity)

Operating Altitude To 2000 m

Water Ingress Non-immersion, protection for damp wipe only

Weight 63.4 kg

Dimensions (W,H,D) 66 cm × 53 cm × 74 cm

Installation GuideThe following section is included for reference purposes only. As standard practice, SGI-DNA will install the BioXp™ System.

CAUTION: Read the entire Installation Guide before unpacking the BioXp™ System. Be sure to read and adhere to the "Safety Warnings" on pages 4 and 5. Contact SGI-DNA immediately if you have any concerns or questions.

System LocationThe BioXp™ 3200 System has a compact footprint and is designed to be used in the typical laboratory environment. Install the instrument in a location that meets the following requirements:

• Lab bench space that is 87 cm (35 inches) wide and 74 cm (29.2 inches) deep to accommodate the instrument and allow for 10 cm of clearance on the sides for proper ventilation.

• Clearance of 79 cm (31 inches) above the lab bench to allow the door to open upward properly

• An available AC power outlet present within 2 meters of the instrument

• Available internet connection through a wired Ethernet connection

7

Setup Precautions

Place the instrument on a clean, dry, flat surface capable of holding the weight of the system.

Allow 10 cm of clearance on both sides of the instrument for proper ventilation.

Make certain that the power cord connection point is free and clear of obstructions so that it is easily accessible.

The intended location must be stable so that the instrument will not move or vibrate excessively when in use.

Avoid dusty areas.

Avoid areas where the instrument would sit in direct sunlight or near a heat source.

Do not set the instrument up where liquids or chemicals are used. Ensure that no liquids are spilled into the instrument.

8

Unpacking, Set up, and Power UpWARNING! Using the BioXp™ System in a manner not specified by SGI-DNA may result in damage to the equipment and/or personal injury.

1. Remove the crate from the BioXp™ System by positioning two people across from each other. Carefully lift the box upward while leaving the instrument stationed on the foam cushions on the pallet as shown in the following illustrations:

2. Remove the foam packaging materials from the top of the instrument.

3. Carefully coordinate lifting the BioXp™ System off of the pallet with at least two people using proper lifting technique:

4. Making certain the path is clear, carry the instrument and place it on the site prepared according to the stipulations outlined in "System Location" on page 6.

5. After removing the power cord from packaging, connect it to the power inlet located on the right rear side panel of the instrument.

6. Check that the power switch located above the power cord inlet is in the “off” position (0).

7. Connect the instrument to a grounded power outlet.

Note: We recommend connecting the instrument to a 1500 VA/900 W uninterruptible power supply.

9

Getting to Know the SystemHardware Overview

Figure 1. Front and side view of the BioXp™ 3200 System.

Figure 2. The BioXp™ 3200 System Deck.

Figure 3. Deck Map.

1– Heated Lid

2– Thermocycler

3– Purification Module

4– Thermal Cover

5– Reagent Chiller

6– Recovery Chiller

7– Reservoir Retainer

8– Strip Well Holder

9– Tip Tray Retainer 1




13– Waste Bin

Instrument Ports and Power Switch

Location Label number Port/Inlet Used to

1 2 3 4 5

1 USB Do not useNote: The USB port is for instrument diagnostics and is for authorized service personnel use only.

2 Ethernet Connect to sgidna.com for system operation

3 Fuse Do not use

4 Power switch

Switch power to the instrument on or off. Note: We recommend leaving the instrument on continuously.

5 Power inlet Power the instrument

Figure 4. Power On/Off switch and communications ports.

10

Starting the BioXp™ SystemEthernet Set UpThe BioXp™ System requires an internet connection. Prior to connecting the instrument to the internet, be sure to test the connection. Connect the provided RJ45 Ethernet Cable to the Ethernet port located on the rear of the unit to an available Ethernet wall jack.

Start the BioXp™ System

1. Turn the instrument on using the power switch located on the side of the instrument.

The BioXp™ System will initiate a self-check process to verify the correct functionality of motion control and other internal systems of the instrument. If the instrument is unable to perform a successful self-check it will alert the user to the problem and will prevent the instrument from operating until the problem has been addressed. The self-check process has been carefully developed to ensure that the BioXp™ System is operating within optimal calibration ranges and that all systems are functioning correctly before initiating a run.

2. After the self-check, one of the following screens is displayed:

If reagents from previous jobs are on the instrument deck, a Remove Materials Screen is displayed. Open the door, remove the materials from previous jobs, and then close the door.

If you are ready to load the instrument and begin a run, refer to the appropriate deck loading instructions for your system run.

11

Quick Start Guide: Performing an Instrument RunAfter installing and setting up the BioXp™ System, the instrument is ready for operation. The following instructions are intended for experienced users. If this is your first time operating the BioXp™ System or if you are unfamiliar with operating the instrument, refer to the detailed instructions on pages 23–29.

BioXp™ Tiles and Libraries Quick StartThe instructions on this page are specific for building BioXp™ Tiles and Libraries. See page 12–14 for instructions for loading the instrument for building BioXp™ Clones.

Each BioXp™ Tiles kit includes Module A (+4°C) and Module B (−20°C). Refer to the Loading Map shipped with your BioXp™ Reagents for the most up-to-date loading instructions.

1. If the door is closed, select "Unlock Door" from the instrument LCD screen and open the door

2. Thaw the DNA Assembly Reagent Plate (at 25°C for 1 hour or on ice for at least 3 hours)

3. Load tips by aligning the tip tray notch with the upper left corner of each Tip Tray Retainer

Load 3 x 50 µL tips Load 1 x 200 µL tips

4. Add a minimum of 12 mL freshly prepared 70% ethanol to the reusable Ethanol Reservoir

Load Ethanol Reservoir in the right-most Reservoir Retainer position of the instrument deck

Note: Do not discard the Ethanol Reservoir after the run; keep for future use

5. Load plates stored at 4°C:

Load the Recovery Plate onto the Recovery Chiller with the notch in the upper left corner

Load the Oligo Vault™ Plate so that the notch is positioned in the upper left corner of the Thermocycler

6. Briefly spin the DNA Purification Strip

Load the black DNA Purification Strip into the #1 position (left-most) with the strip pinhole closest to the front of the instrument

7. Secure strips with the spring-loaded arms while holding strips securely in place

8. Spin the thawed DNA Assembly Reagent Plate for 1 minute at 500 rpm. Visually inspect the wells to ensure that they are completely thawed before loading the plate.

Load DNA Assembly Reagent Plate onto Reagent Chiller, notch in the lower left corner

Note: Be certain that the plate is properly seated within the chiller and that the barcode is on the right

9. Refer to the photo in the lower left panel below. Confirm that all components are securely seated. Close the door.

10. After the deck inspection, press Start Now or Delay Start (no more than 2 hours) to begin the run.

Loaded Deck at Beginning of Run Deck at Run Completion

Oligo Vault™

Thermal Cover DNA Assembly Reagent Plate

Recovery Plate

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Oligo Vault™Recovery Plate

Location of BioXp™ Tiles after the runAfter the instrument run, BioXp™ final products are located in Wells A1–H4 of the Recovery Plate within the Thermocycler. BioXp™ Tiles and Libraries are provided in 45 μL TE Buffer. The expected yield is >200 ng of purified, linear DNA. If you will not be using the final products immediately, seal the Recovery Plate and store at 2°C to 8°C for up to one week or at −20°C for up to one year.

12

BioXp™ Cloning Quick StartThe following instructions are intended for experienced users only. For additional instructions, see:

• The BioXp™ Custom Cloning Vector Preparation Guide available at sgidna.com.

• Detailed loading instructions on pages 23–29.

Guidelines for preparing the BioXp™ custom vector strip

1. After you have prepped your vector according to the protocols and guidelines in the BioXp™ Custom Cloning Vector Preparation Guide available at sgidna.com, adjust the prepared vector concentration according to the table:

Vector Size (kb) Concentration (ng/µL)

3–5 15–20

5–7 20–25

7–9 25–30

9–12 30–35

2. Determine the volume of prepared linear vector you will add to wells A, C, E, and G of a BioXp™ Vector strip.

Note: Volume recommendations are subject to change. Please contact us at [email protected] for more information.

Number of BioXp™ Cloning Reactions Volume/well

≤16 Reactions 12 µL

>16 Reactions 18 µL

3. Add 12 or 18 µL of prepared linear vector to wells A, C, E and G of a BioXp™ Vector strip. Do not seal the strip. Ensure that no air bubbles have been introduced and that the resuspended vector is at the bottom of the strip wells. Load the filled custom strip on the deck with the pinhole closest to the front of the instrument. A C E GB D F H

pinhole

EXAMPLE: For 8 cloning reactions, adjust the volume of a 10 kb vector to a final concentration of 30–35 ng/µL. Add 12 µL of the linearized, prepared vector to the four strip wells (A, C, E and G).

Total amount of required vector = 1.44 to 1.68 µg.

13

BioXp™ Cloning Quick Start: Loading the instrument

Each BioXp™ Cloning kit includes Module A (+4°C), Module B (−20°C), and Module C (−20°C).

1. If the door is closed, select "Unlock Door" from the instrument touch screen and open the door

2. Thaw −20°C components as directed below:

• DNA Assembly Reagent Plate (at 25°C for 1 hour or on ice for at least 3 hours)

• GA Cloning Strip and Vector Strip (30 minutes on ice)

3. Load tips by aligning the tip tray notch with the upper left corner of each Tip Tray Retainer

Load 3 x 50 µL tips


4. Add a minimum of 12 mL freshly prepared 70% ethanol to the reusable Ethanol Reservoir

Load Ethanol Reservoir in the right-most Reservoir Retainer position of the instrument deck

Note: Do not discard the Ethanol Reservoir after the run; keep for future use


Load the Recovery Plate onto the Recovery Chiller with the notch in the upper left corner

Load the Oligo Vault™ Plate into the Thermocycler with the notch in the upper left corner

6. Briefly spin the strips. Visually inspect the wells to ensure that they are completely thawed. Load in the order listed and shown below, with the strip pinhole closest to instrument front

Load the black DNA Purification Strip into position #1

Leave position #2 empty

Load the purple GA Cloning Strip into position #3

Load the red pUCGA 1.0 or Custom Vector Strip into position #4

7. Secure strips with spring-loaded arms while holding the strips in place.

8. Spin the thawed DNA Assembly Reagent Plate for 1 minute at 500 rpm. Visually inspect the wells to ensure that they are completely thawed.

Load DNA Assembly Reagent Plate onto Reagent Chiller, notch in the lower left corner and barcode on the right

Note: Be certain that the plate is properly seated within the chiller.

9. Refer to the photo in the lower left panel below. Confirm that components are securely seated. Close the door.

10. After the deck inspection ends, press Start Now or Delay Start (no more than 2 hours) to begin the run.

Loaded deck at beginning of run

Oligo Vault™

Thermal CoverDNA Assembly Reagent Plate Recovery

Plate

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14

After the cloning run: final DNA product location

Deck at run completion

Recovery Plate Oligo Vault™

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keep

Items to keep

DNA product Located within Storage instructions

Cloning Reaction (wells A1–H4) Recovery Plate

Seal and store at 2°C to 8°C for up to one week or at −20°C for up to one year.Uncloned Tiles (wells A5–H8)

Oligo Vault™ Plate (foil covered)

Ethanol Reservoir (empty and rinse for next use)

Recommendations

After the BioXp™ run is complete, cloning reactions are ready for transformation. We recommend diluting cloning reactions 1:2 with molecular biology grade water before transformation.

Analyze Tiles– We recommend evaluating the success of the assembly reaction by running a gel containing the uncloned BioXp™ Tiles from the Oligo Vault™ Plate before transforming clones.

Transformation– We recommend using E. cloni 10G Chemically Competent Cells (Lucigen Cat. No. 60107, free samples available at Lucigen.com) or TransforMax™ EPI300™ Electrocompetent E. coli (Lucigen Cat. No. EC300110). If other cells are used, use competent cells with a transformation efficiency ≥1 × 109 CFU/μg pUC19.

15

Operational Features of the BioXp™ SystemDelay Start

The Delay Start function is a useful tool enabling you to schedule instrument runs at your convenience. Immediately after a Delay Start is scheduled (before the delay time period elapses), the instrument initiates reagent mixing from the Reagent Strips and the Reagent Plate. Therefore, ensure that the Reagent Strips and Reagent Plate have thawed completely before initiating a Delay Start. If you decide to cancel a Delay Start, consider that you may need to replace the Reagent Strips and Reagent Plate and run the job at a later date since the master mixes created from combining the contents of the Reagent Strips and the Reagent Plate are thermolabile.

To perform a Delay Start:

1. After pressing Delay Start on the Job Initiation Screen, the system will display a Delay Timer Screen:

2. Use the arrows to set the delay start time and press OK.

Note: We recommend using the Delay Start feature for short periods of time (less than 2 hours) as necessary.

3. After the delay time has been scheduled, the system will prepare reagents for the run. The system displays a Delay Start Screen with a timer showing the length of time left until the run begins (in hours : minutes : seconds) and the expected time of job completion (also in hours : minutes : seconds):

4. After the system has prepared the reagents for the run, a new countdown screen is displayed. You may opt to bypass the delay and start the run immediately by pressing Start Now. If no action is taken, the instrument run will begin when the timer reaches 0:00.

16

Tracking Progress During a Run

Time to CompletionDuring a run, the instrument will display the projected time remaining for the run (in hours : minutes : seconds) and the estimated Time of Completion, as shown in the following illustration.

Ways to Track ProgressYou can track the progress of your reaction in a number of ways:

Parameter Definition

Time to Completion The system estimates the time at which the run will be completed.

Time Remaining The system estimates the time remaining for completion.

Visual Progress The system provides a progress bar through the various steps to completion. Note: The scale of each step is not linear. (i.e. Step 1 requires much more time than Step 4 but both steps are each depicted with a similar sized time lapse bar).

Color IndicatorsThe internal colored lights change during the run process. The colors indicate the status of the process taking place. The background color of the screen changes to indicate the following:

Touchscreen Background Color Indication

Blue Normal operation: run set up or run in progress

Yellow System check or prompt requiring user attention

Red Error message

Green Processing completed

17

Canceling a Job During Delay Start

To cancel a job after initiating a Delay Start, press Cancel Job .

The system will display a screen reminding you that materials may have already been compounded, rendering reagents unusable at a future time.

Press To

NO (keep running) Continue with the delay start and instrument run

YES (stop running) Cancel the delay start and abort the job

Canceling a Job During a RunIMPORTANT! You will not be able to reuse reagents if you cancel a job after the run has initiated and the Reagent Plate Seal has been broken. Do not cancel a run unless absolutely necessary.

1. If you need to cancel a job during a run, slide your finger to the right across the bar on the bottom of the Display Screen during a Run.

Slide to cancel

2. After sliding to cancel, the system will display a Confirmation To Cancel screen. Press YES Cancel Job to end the run or No to continue the run.

If you press YES Cancel Job, the instrument will display a Canceling Job screen until the job cancel is complete.

3. When prompted, remove all materials and used or partially used consumables from the instrument deck:

Shutting down the BioXp™ SystemWe recommend leaving the instrument on continuously. Only turn the instrument off when necessary, such as when moving the instrument to another location. If the instrument must be turned off, toggle the power switch located on the side of the instrument (near the power cord) to the OFF position.

18

Configuring the BioXp™ SystemFrom the Configuration screen, you may change the clock.

If you press... The System Displays Allowing you to set the clock

Set Date and Time • Check Automatically get time to set the clock automatically. Then press OK.OR

• Use the up and down arrows to set the time and date manually. Then press OK.Notes:

• Time is displayed in hours : minutes : seconds and the Date is displayed in Month/Day/Year.

• Check Observe Daylight Saving Time for automatic clock updates to DST.

19

Instrument TroubleshootingGeneral Instrument Troubleshooting

Issue Possible Solutions and Recommendations

The touch screen does not light up • Confirm that the power switch is in the "ON" position.• Confirm that the instrument is plugged into a working electrical outlet.• Verify that the plug is securely connected to the side of the instrument.• Contact SGI-DNA Technical Services, 1-855-474-4362.

Power failure occurs during the run• Contact SGI-DNA Technical Services, 1-855-474-4362.

Difficulty operating the touchscreen

Deck InspectionAfter you load the deck and close the instrument door for a system run, the instrument performs a deck inspection, checking the location and orientation of the Recovery Plate, the Ethanol Trough, the strip handles, the 8-well strips, tips, and the system robotics.

If any anomalies are detected, the instrument displays a deck inspection failure message, similar to the screen shown on the right. If the system displays a deck inspection failure message, see the following table for recommendations.


Deck inspection detected anomalies with one (or more) of the following:• Plate found in purification nest• Recovery Plate not found or is rotated• Ethanol Trough is missing or

misplaced• Strip handle not closed

1. Touch Stop and Fix 2. Open the door and correct the deck inspection issue.3. After fixing the error, close the door to initiate a second deck

inspection.4. If the deck inspection error message persists more than once after you

have corrected any issues, please call SGI-DNA Customer Service at 1-855-474-4362. You may proceed with the instrument run by touching Ignore and Continue

Error MessagesIf you observe a red-background error screen during operation, follow the screen instructions. If directed or if the problem persists, make a note of the screen and Call SGI-DNA Customer Service at 1-855-474-4362. Additional information is included in the following table.


Problem preventing the system from running the job

• May be caused by a barcode reading error. Open the door and confirm that all plates are properly seated in their respective retainers in the correct configuration, then press Retry.

• Press Configuration and follow the on screen instructions.

20

Cleaning and MaintenanceCleaning Instructions

Clean the exterior of the BioXp™ System with standard cleaners and a damp cloth.

Clean the internal surfaces periodically using a mild detergent or 70% ethanol. Spray the cleaner onto a cloth and then use the damp cloth to wipe instrument surfaces. Do not spray any cleaners directly into the instrument.

Do not use abrasive or caustic cleaners on internal or external instrument surfaces.

SpillsTake every precaution to ensure that no liquids are spilled into the instrument. Wipe up accidental spills in and around the instrument immediately. If ethanol spills, dispose of ethanol-saturated towels according to your institutional guidelines.

In the event of hazardous material spillage• In the event of spillage of a hazardous material, the BioXp™ System user is responsible for decontamination of

the hazardous material on or inside the instrument.

• Before using a cleaning or decontamination method other than a recommended cleaner, contact SGI-DNA to verify that the proposed decontamination method will not damage the equipment.

CalibrationCalibration of the motion control system is performed by the manufacturer; no periodic calibration is required. The instrument will perform a self-check each day. A log of the self check is stored on the system and may be used by SGI-DNA Service Technicians should any issue arise.

Preventative MaintenanceThe BioXp™ System does not require any preventative maintenance besides normal cleaning.

RepairEach BioXp™ System has been manufactured and calibrated to specifications. Do not attempt to disassemble any part of the system. Any such action will void the product warranty. In the event you should experience a product failure, please call SGI-DNA Technical Support at 855-474-4362.

Recommended Separation DistancesEquipment and Systems that are NOT Life-supportingThe BioXp™ System is intended for use in the electromagnetic environment specified below. The customer or user of the BioXp™ System should ensure that it is used in such an environment. The BioXp™ System is intended for use in the electromagnetic environment in which radiated disturbances are controlled. The customer or user of the BioXp™ System can help prevent electromagnetic interference by maintaining a minimum distance between portable and mobile RF Communications Equipment and the BioXp™ System as recommended below, according to the maximum output power of the communications equipment.

Rated maximum outputpower of transmitter

W

Separation distance according to frequency of transmitterm

150 kHz to 80 MHzd = [3.5]√P V1

80 MHz to 800 MHzd = [3.5]√P E1

800 MHz to 2.5 GHzd = [3.5]√P E1

0.01 0.12 0.12 0.23

0.1 0.38 0.38 0.73

1 1.2 1.2 2.3

10 3.8 3.8 7.3

100 12 12 23

For transmitters rated at a maximum output power not listed above, the recommended separation distance d in meters (m) can be estimated using the equation applicable to the frequency of the transmitter, where P is the maximum output power rating of the transmitter in watts (W) according to the transmitter manufacturer.

NOTE 1: At 80 MHz and 800 MHz, the separation distance for the higher frequency range applies.NOTE 2: These guidelines may not apply in all situations. Electromagnetic propagation is affected by absorption and reflection from structures, objects, and people.

21

Equipment DisposalThis product has parts that contain a small amount of mercury and lead in some components. Disposal of these materials may be regulated in your community due to environmental considerations. Follow local regulations for disposal of equipment or equipment accessories at the end of their life.

WEEE ComplianceThe BioXp™ System complies with the European Union's Waste and Electrical and Electronic Directive. For disposal or recycling information, please contact your local authorities or the electronic industry alliance: www.eiae.org.

22

Regulatory StatementsFCC Compliance StatementThis equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference in a commercial or medical installation. This equipment generates, uses and can radiate radio frequency energy and, if not installed and used in accordance with the instructions, may cause harmful interference to radio or television reception. However, there is no guarantee that interference will not occur in a particular installation. If this equipment does cause interference to radio and television reception, which can be determined by turning the equipment off and on, the user is encouraged to try to correct the interference by one or more of the following measures:• Reorient or relocate the receiving antenna• Increase the separation between the equipment and the receiver• Connect the equipment into an outlet on a circuit different from that to which the receiver is connected• Consult the dealer or an experienced radio/TV technician for help

The connection of a non-shielded equipment interface cable to this equipment will invalidate the FCC Certification of this device and may cause interference levels which exceed the limits established by the FCC for this equipment. It is the responsibility of the user to obtain and use a shielded equipment interface cable with this device. If this equipment has more than one interface connector, do not leave cables connected to unused interfaces. Changes or modifications not expressly approved by the manufacturer could void the user’s authority to operate the equipment.

Portable and mobile RF communication devices such as cellular phones can adversely affect medical equipment.

Guidance and Manufacturer’s Declaration: EmissionsAll equipment and systems

Emission tests Compliance Electromagnetic environment guidance

RF emissions CISPR 11 Group 1 The BioXp™ System uses RF energy only for its internal function. Therefore, its RF emissions are very low and are not likely to cause any interference in nearby electronic equipment.

RF emission CISPR 11 Class A

The BioXp™ System is suitable for use in all commercial establishments.Harmonic emission IEC 61000-3-2 Class A

Voltage fluctuations IEC 61000-3-3 Complies

Guidance and Manufacturer’s Declaration: ImmunityAll equipment and systemsThe BioXp™ System is a class A device and is intended for use in the electromagnetic environment specified below.

The customer or user of the BioXp™ System should ensure that it is used in such an environment.

Immunity test IEC 60601 test level Compliance level Electromagnetic environment guidance

Electrostatic discharge (ESD) IEC 61000-4-2

±6 kV contact±8 kV air

±6 kV contact±8 kV air

Floors should be wood, concrete, or ceramic tile. If floors are covered with synthetic material, the relative humidity should be at least 30%.

Electrical fasttransient/burstIEC 61000-4-4

±2 kV for power supply lines

±1 kV for input/output lines

±2 kV for power supply lines

±1 kV for input/output lines

Mains power quality should be that of a typical commercial or laboratory environment.

SurgeIEC 61000-4-5

±1 kV differential mode±2 kV common mode

±1 kV differential mode±2 kV common mode

Mains power quality should be that of a typical commercial or laboratory environment.

Voltage dips, short interruptions, and voltage variations on power supply input lines IEC 61000-4-11

<5% UT (>95% dip in UT) for 0.5 Cycle40% UT (60% dip in UT) for 5 Cycles70% UT (30% dip in UT) for 25 Cycles<5% UT (>95% dip in UT) for 5 sec.

<5% UT (>95% dip in UT) for 0.5 Cycle40% UT (60% dip in UT) for 5 Cycles 70% UT(30% dip in UT) for 25 Cycles<5% UT (>95% dip in UT) for 5 sec.

Mains power quality should be that of a typical commercial or laboratory environment. If the user of

the BioXp™ System requires continued operation during power mains interruptions, it is recommended that the instrument be powered from an uninterrupted power supply or a battery.

Power frequency(50/60 Hz) magnetic field IEC 61000-4-8

3 A/m 3 A/mPower frequency magnetic fields should be at levels characteristic of a typical location in a typical commercial or laboratory environment.

NOTE: UT is the AC mains voltage prior to application of the test level.

23

BioXp™ Tiles, Libraries, and Clones

Product Ordering InformationTo order reagents for gene construction, log into your account, and upload your DNA fragments-of-interest to the SGI-DNA secure website: https://www.sgidna.com.

OverviewReceiving an Order

Your reagent shipment for building BioXp™ products contains two boxes (for BioXp™ Tiles and Libraries) or three boxes (for BioXp™ Clones). Upon receipt, confirm items by matching the Stock Keeping Unit (SKU) printed on each item with the SKU listed on your accompanying Job-Specific BioXp™ Product Map. All items are unique to a specific job; use only as indicated.

BioXp™ +4°C Module (included with all products)

Component Quantity Storage Temperature

Oligo Vault™ Plate 1 plate

Store at 4°CDNA Purification Strip (black) 1 strip

Recovery Plate 1 plate

Product Map and Reagent List 1 document N/A

BioXp™ −20°C Module (included with all products)


DNA Assembly Reagent Plate 1 plate Store at −20°C

BioXp™ Cloning Reagents (included for BioXp™ Clones orders only)


GA Cloning Strip (purple) 1 strip

Store at −20°C

Vector Strip (red) 1 strip

Required materials (not included in the product order)

From SGI-DNA

Accessory products Quantity Cat. No.

BioXp™ 50 µL Natural Tips* 1 Case of 24 Racks 12020 CS

BioXp™ 200 µL Conductive Black Tips* 1 Case of 24 Racks 12021 CS

*BioXp™ Tips have been standardized for use on the instrument. It is critical to the success of BioXp™ system runs that these are the only tips used with the BioXp™ instrument.

User supplied reagents

Item Notes Recommended supplier and Catalog Number

Fresh 70% Ethanol You will need a minimum of 12 mL per run —

(Optional) High Transformation Efficiency Competent Cells (≥ 1 × 109 CFU/μg pUC19)

We recommend chemically competent E. cloni® 10G cells or TransforMax™ EPI300™ Electrocompetent E. coli

Lucigen Cat. No. 60107 orLucigen Cat. No. EC300110

Uninterruptible Power Supply 1500VA / 900W —

User supplied equipment

Item Notes Recommended supplier and Catalog number

Bio360™ Minifuge or comparable

Must be capable of 8-well strip centrifugation SGI-DNA Cat. No. SGI-C1008-B

(Optional) Electroporation System

Necessary if using electrocompetent cells

Gene Pulser Xcell™ Electroporation System: Bio-Rad Cat. No. 1652660

SpecificationsThe BioXp™ 3200 System is designed to rapidly synthesize high-quality, linear DNA fragments from custom designed oligonucleotide pools and reagents across a meaningful section of the complexity continuum. A capacity of 32 DNA fragments (Tiles), ranging in size from 350 bp to 1.8 kb, can be assembled with high fidelity. DNA fragments synthesized by the BioXp™ 3200 System are ready for off-line sequencing QC, further assembly into larger fragments, cloning into vectors with compatible ends and other common downstream applications.

Construct Specifications

Specification BioXp™ Tiles BioXp™ Degenerate Library Tiles BioXp™ Clones

Size Range 350–1800 bp 300–800 bp (recommended) 350–1800 bp

GC% Complexity

Complexity Size range20–70% GC 350 bp – 1.0 kb40–60% GC 350 bp – 1.8 kb

20 – 70% (within recommended size range)

Complexity Size range20–70% GC 350 bp – 1.0 kb40–60% GC 350 bp – 1.8 kb

Composition A, T, C or G residue IUPAC code residues A, T, C or G residue

Yield >200 ng of purified, linear dsDNA in 45 µL TE Buffer

>200 ng of purified, linear dsDNA library in 45 µL TE Buffer

~ 20 µL of cloned product (± 2 µL) and~ 40 µL of uncloned Tiles (± 2 µL)

Format 1 to 32 reactions in a 96-well plate format

1 to 32 reactions in a 96-well plate format

1 to 32 reactions in a 96-well plate format

24

Consumables and ReagentsConsumablesImage Item Remarks

50 µL Tip Tray

(3 Trays per run)

Plate notch is located in the upper left hand corner of the tray, near the well A1 position

200 µL Tip Tray

(1 Tray per run)

Plate notch is located in the upper left hand corner of the tray, near the well A1 position

Recovery Plate

(1 Plate per run)

Plate notch is located in the upper left hand corner of the plate, near the well A1 position

Reusable Instrument Components

Ethanol Reservoir Reusable

Reagent and Recovery Plate Thermal Covers (for the Cover Stands)

Placed during initial instrument set upThe instrument returns thermal covers to proper locations at the end of each run

Reagents

Notch

Oligo Vault™ PlateThe Oligo Vault™ carrier notch is in the upper left hand corner of the plate, near the well A1 positionHoused in a Carrier Tray

Notch

DNA Assembly Reagent Plate

The DNA Assembly Reagent Plate notch is in the lower left hand corner of the plate

DNA Purification Strip Black Strip Color

Pinhole at one end of the strip

GA Cloning Strip

(included with BioXp™ Clones orders only)

Purple Strip ColorPinhole at one end of the strip

pUCGA 1.0 Stripor

Custom Vector Strip

(included with BioXp™ Clones orders only)

Red Strip ColorPinhole at one end of the strip

70% ethanol for Ethanol Reservoir

Prepare fresh 70% ethanol User supplied (not included with reagents)

IMPORTANT: Be sure to match all reagents to your BioXp™ System order. Each system run is accomplished from a matched set of reagent components.

25

26

Instrument Operation Preparing for a BioXp™ 3200 System run

Thaw ReagentsRemove reagents from storage and thaw according to the following instructions:

Component* Thawing temperature and duration


Thaw at 25°C for 1 hour or on ice for at least 3 hours

GA Cloning Strip (for building BioXp™ Clones only) Thaw on ice for 30 minutes

Note: Do not thaw at room temperatureVector Strip (for building BioXp™ Clones only)

* Contents in plates and strips can be dispersed during shipping and handling. Pulse spin the plates and strips after thawing.

Fill and load the Ethanol Reservoir

1. Prepare 20 mL of fresh 70% ethanol by adding 14 mL of 200 proof ethanol to 6 mL of nuclease-free water.

Note: Use fresh ethanol for best results.

2. Add ≥12 mL fresh 70% ethanol to the reusable Ethanol Reservoir.

Note: Do not add ethanol to the reservoir while the reservoir is in the retainer on the instrument deck. Always remove the reservoir to a lab bench away from the instrument before filling to prevent any accidental spillage from seeping onto the instrument.

3. Secure the reservoir lid onto the Ethanol Reservoir and place in the right-most side of the Reservoir Retainer on the instrument deck as shown.

Note: When loading the reservoir onto the instrument deck, ensure that the reservoir fits snugly within the retainer and that the tabs of the reservoir are seated properly within the retainer groove.

Centrifuge strips and plates

After the strips and DNA Assembly Reagent Plate have thawed, briefly spin the following components to ensure that the contents are at the bottom of the wells:

• Spin the thawed DNA Assembly Reagent Plate for 1 minute at 500 rpm

• Pulse spin the thawed GA Cloning and Vector Strips (for building BioXp™ Clones only)

• Pulse spin the DNA Purification Strip

27

Load the instrument 1. If the door is closed, select "Unlock Door" from the instrument touch screen and open the door.

2. Load tips by aligning the tip tray notch with the upper left corner of each Tip Tray Retainer.



3. Confirm that you have added a minimum of 12 mL freshly prepared 70% ethanol to the reusable Ethanol Reservoir in the right-most Reservoir Retainer position of the instrument deck.


Load Recovery Plate onto Recovery Chiller with the notch in the upper left corner

Load Oligo Vault™ Plate into Thermocycler with the notch in the upper left corner

5. Collect strip tube contents at the bottom of the wells by performing a quick spin or centrifugation prior to loading. Visually inspect the wells to ensure that they are completely thawed before loading the strips.

Load black DNA Purification Strip – #1 position (left-most), with pinhole near front of instrument

Leave position #2 empty

Load the purple GA Cloning Strip into position #3 (for building BioXp™ Clones only)

Load the red pUCGA 1.0 or Custom Vector Strip into position #4 (for building BioXp™ Clones only)

6. Secure strips with the spring-loaded arms while holding strips securely in place with your fingers.

7. Spin the thawed DNA Assembly Reagent Plate for 1 minute at 500 rpm. Visually inspect the wells to ensure that they are completely thawed before loading the plate.

Load DNA Assembly Reagent Plate onto Reagent Chiller

Note: Be certain that the plate is properly seated within the chiller.

8. Confirm that all components are securely seated and close the door. Use the following illustration as a reference for a properly and completely loaded instrument deck.

Loaded deck at the beginning of a run (pUCGA Cloning is shown)

Oligo Vault™

Thermal CoverDNA Assembly Reagent Plate Recovery

Plate

Etha

nol

Rese

rvoi

r



blac

k st

rip

purp

le st

ripre

d st

rip

28

Begin the Run1. Close the door.

Note: You will hear the door latch lock and the door will remain locked throughout the instrument run. Do not open the door during a run.

2. The system reads the job barcode and displays a Job Initiation Screen:

Press... To...

Begin the run.

Cancel the run.

Delay the run start time (up to 2 hours).

During the RunAn instrument run generating BioXp™ Tiles or Clones generally takes 12–17 hours. Library runs take approximately 6–8 hours.

After the RunRemoving Materials from the InstrumentWhen the run is complete, the system displays the Job Complete Screen:

1. Touch Unlock Door to open the door.

Note: Expect a delay (< ~1 minute) after pressing Unlock Door before the door is unlocked and ready to be opened.

2. Remove all applicable materials. Final DNA products are located in the Recovery Plate, which has been moved to the Thermocycler at the end of the run. Each reaction well (Wells A1–H4) contains assembled Tiles or Libraries in 45 μL TE Buffer, or clones in approximately 20 μL volume.

3. Properly discard or store each item (refer to the table on the following page for instructions).

29

Final DNA Product LocationDuring the run, materials are moved from their original loading position. After completion of the instrument run, final DNA products are in the Recovery Plate located within the Thermocycler. You may store BioXp™ Tiles, Libraries, and Clones by applying a seal to the Recovery Plate and placing the plate at 4°C for short-term (1 week) or at −20°C for long-term storage. We recommend sealing the Recovery Plate with an adhesive film (e.g., Nunc™ Sealing Plates, Thermo Fisher Cat. No. 236370) prior to storage. Properly discard or store each of the following items.

ItemDeck

location at end of run

Save or Discard? What should I do with each item?

Recovery Plate Thermocycler Save

The Recovery Plate, located within the Thermocycler, contains the BioXp™ Tiles, Libraries, or Clones.Seal the plate and store at 2°C to 8°C for up to one week or at −20°C for up to one year.

Note: Final DNA products (BioXp™ Tiles, Libraries, or Clones) are located in wells A1–H4.

Oligo Vault™ Plate

Purification Module

Save for BioXp™ Clones

Discard for BioXp™ Tiles and

Libraries

For BioXp™ Clones, you may save the foil-covered Oligo Vault™ Plate for troubleshooting. All other components of the Oligo Vault™ may be discarded. The Oligo Vault™ Plate contains uncloned BioXp™ Tiles (in wells A5–H8), which you may choose to save for further analysis or other applications. Refer to your job-specific BioXp™ Product Map for the order and location of the Tiles within the plate. We recommend aspirating the BioXp™ Tiles and transferring the Tiles to a clean 96-well plate for optimal DNA preservation. Seal and store the plate at 2–8°C for up to one week or at −20°C for up to one year.

For BioXp™ Tiles and Libraries, you may discard the Oligo Vault™ Plate


Reagent Chiller Discard —

DNA Purification Strip

Strip Well Holder Discard —

Ethanol Reservoir Reservoir Rack Save

Empty and discard remaining ethanol according to the appropriate guidelines for hazardous chemical waste. Rinse the Ethanol Reservoir with water and dry the Reservoir so that it will be ready for future use.

Waste bin — Save Empty after use and dispose of used pipette tips in an appropriate waste container.

Tip Trays Tip Tray Receptacles Discard You may consolidate and save unused tips for future runs

if desired.

30

Analyzing BioXp™ Tiles, Libraries, and ClonesGel Electrophoresis

BioXp™ Tiles and Libraries BioXp™ Clones

After removing BioXp™ Tiles from the instrument deck, the Tiles are ready for use. We recommend analyzing the BioXp™ Tiles by gel electrophoresis to confirm the presence of a single band of the expected size before proceeding to downstream applications of your choice (e.g., cloning, PCR amplification, and sequencing).

Analyze the uncloned BioXp™ Tiles (located in the Oligo Vault) by gel electrophoresis to gauge the success of the assembly reaction. We recommend confirming successful Tile assembly before transforming the clones or initiating other downstream applications of the clones. Cloning results will be compromised if Tiles failed to assemble.

Instructions for Tiles and Libraries 1. Run a 1–2% agarose gel containing the Tiles.

2. Evaluate the gel (see the following sections for more information).

Interpreting the gel: Tiles and LibrariesIf you observe results similar to the image shown in Figure 5A with prominent single bands of the expected size, proceed directly to downstream applications of your choice.If you observe the presence of by-products or weak bands (Figure 5B), you should gel purify the fragment of interest before proceeding to downstream analysis.

Instructions for ClonesLinear BioXp™ DNA Tiles are located in wells A5–H8 of the Oligo Vault™ Plate.

1. Pull ~5 μL of the suspended BioXp™ Tiles away from the purification beads for gel analysis. Note: Liquid evaporation is common in the Oligo Vault™ Plate at this stage. You may need to add 10 μL ddH2O to reconstitute the DNA.Important: To save BioXp™ Tiles, move into a clean plate or tube to remove from the purification beads before storing.

2. Run a 1–2% agarose gel containing the Tiles.3. Evaluate the gel (see the following sections for more

information).

Interpreting the gel: ClonesProminent single bands of the correct size (see Figure 5A) typically correlate with high cloning efficiencies. Proceed to transformation of the corresponding clone.Weak bands or the presence of secondary bands (see Figure 5B) may correlate with lower cloning efficiencies. If you choose to proceed with transformation, you may need to screen additional colonies. We recommend screening by colony PCR prior to sequence verification in this case. Alternatively, you may choose to gel purify your fragment of interest and manually clone the fragment.

Expected ResultsTo confirm the presence of your assembled product, perform agarose gel electrophoresis. The following image (Figure 5A) shows an example of a completed run with the assembled fragment of interest clearly distinguishable, noted in the image. Notice the absence of off-target assemblies and/or intermediate by-products in the image, which is indicative of an efficient BioXp™ System run. If you observe similar results, proceed directly to downstream applications of your choice. Figure 5B shows an example of a completed run with the fragment of interest as well as the presence of intermediate by-products. Although the presence of by-products indicates that the run was not optimally efficient, you may still gel purify the fragment of interest and proceed to downstream analysis.

1 2 L 3 L

1.5 kb Tile

10 kb

1 kb

0.5 kb

1.5 kb Tile

by-products

10 kb

1 kb

0.5 kb

Expected result

Presence of by-products

A B

Figure 5. Gel analysis to gauge efficiency of Tile build. A 1.5 kb gene was assembled with the BioXp™ System and analyzed using electrophoresis on a 2% agarose gel containing ethidium bromide. Lanes 1 and 2 (Panel A) and Lane 3 (Panel B) contain 2 µL of final product from 3 different reactions. The far right lane in each gel contains 500 ng of 2-Log DNA ladder (New England BioLabs).

The next sections describe how to analyze your DNA products and the next steps you will take for downstream applications.• For BioXp™ Tiles or Libraries, see "Next Steps– BioXp™ Tiles and Libraries" on pages 31–33.• For BioXp™ Clones, see "Next Steps– BioXp™ Clones" on pages 34–35.

31

Next Steps– BioXp™ Tiles and LibrariesThe following sections are instructions for BioXp™ Tiles and Libraries. For BioXp™ Clones, see "Next Steps– BioXp™ Clones" on pages 34–35.

After an instrument run, perform gel electrophoresis (described on page 30) of BioXp™ Tiles and Libraries before proceeding with the following instructions.

QuantificationTo quantify BioXp™ Tiles in each applicable well of the Recovery Plate, use 1–2 µL in a Qubit® 2.0 Fluorometer or comparable device used for quantifying DNA.

Cloning BioXp™ Tiles

BioXp™ Tiles, True ends (including Degenerate Library Tiles)BioXp™ Tiles are DNA fragments synthesized using the BioXp™ System. BioXp™ Tiles, True ends (including Degenerate Library Tiles) are built without any adapters or other modifications to the submitted sequence and can be cloned into any vector using your preferred method. BioXp™ Tiles are ready for use after running a gel to confirm the presence of a single species of DNA of the expected size. BioXp™ Tiles, True ends can be used in any number of cloning applications.

BioXp™ Tiles, GA endsBioXp™ Tiles, GA ends are Tiles built with specific GA end sequences that are homologous with terminal sequence of the SGI-DNA pUCGA 1.0 vector (Cat No. GA1050-100), enabling streamlined assembly using the Gibson Assembly® method. For more information, please contact Customer Service at [email protected].

Multi-fragment AssemblyMultiple BioXp™ Tiles, True ends may be assembled together to build larger constructs. Multi-fragment assembly requires designing overlapping ends between the fragments prior to synthesis. BioXp™ Tiles that have been designed accordingly may be assembled using the Gibson Assembly® HiFi 1-Step Kit or the Gibson Assembly® Ultra Kit.

For assistance with the proper design of constructs for efficient hierarchical assembly, contact SGI-DNA at [email protected] or 1-855-474-4362.

32

PCR Amplification (Optional)If you require additional material for your downstream application, you may amplify your assembled product. We recommend the following components and conditions.

1. For each assembly product, prepare the following reaction mixture in a thin-walled PCR tube:

Component Final Concentration Volume

Final Assembly Product template ≤0.1 µg/µL χ μL

Amplification Primers† (25 µM‡) 0.5 µM 1 μL

Q5® High-Fidelity 2X Master Mix† (NEB, Catalog number M0492)

1X 12.5 μL

Nuclease-free water† — 11.5–χ μL

Total 25 μL

† User-supplied‡ Each primer is 12.5 µM

2. Transfer tubes to a thermocycler. Program and start the thermocycler using the following conditions:

Initial Denaturation 98°C 1 minute 1 Cycle

Amplification 98°C 10−30 seconds

30 Cycles60°C 30 seconds

68–72°C 30–45 seconds

Final Extension 72°C 5 minutes 1 Cycle

Hold 4°C as necessary 1 Cycle

33

Colony Screening (Optional)To reduce the number of colonies you will analyze by sequencing, we recommend pre-screening colonies with colony PCR or restriction enzyme digestion.

For colony PCR procedure:

1. Combine the following PCR components on ice:

Component* Final Concentration Volume

Amplification Primers 200 nM each (Fwd and Rev) χ μL

OneTaq® Quick-Load 2X Master Mix with Standard Buffer (NEB, Catalog number M0486)

1X 25 μL

Nuclease-free water — 25–χ μL

Total 50 μL

* Combine all PCR components in a cocktail with a total volume 10% greater than that required for the total number of PCR assays to be performed. After combining all components, aliquot 50 μL of the cocktail into individual PCR tubes.

2. Use a sterile toothpick or a P200 pipette tip to pick a colony.

3. Dip the toothpick or pipette tip into the PCR tube containing the PCR cocktail and gently swirl for a couple of seconds.

4. Transfer the toothpick or pipette tip into liquid growth media containing appropriate selective antibiotics and culture overnight.

5. Start the PCR reaction:

Initial Denaturation 94°C 2 minutes 1 Cycle

Amplification 94°C 30 seconds

30 CyclesPrimer Tm 30 seconds

68°C 1 minute/kb

Final Extension 68°C 10 minutes 1 Cycle


6. Load ~5 μL onto an agarose gel for analysis.

7. Identify full-length clones by determining properly sized amplicons.

8. Proceed to "Full-length Clone (EFC) Analysis" on page 36.

34

Next Steps– BioXp™ ClonesAfter an instrument run, perform gel electrophoresis (described on page 30) of BioXp™ Tiles and Libraries before proceeding with the following instructions.

TransformationClones are located in wells A1–H4 of the Recovery Plate.

We recommend diluting cloning reactions 1:2 with molecular biology grade water before transformation.We recommend using the following protocol with E. cloni 10G chemically competent cells (Lucigen Cat. No. 60107). If you are using competent cells other than Lucigen 10G cells, follow the transformation protocol provided with your competent cells. Use competent cells with a transformation efficiency ≥1 × 109 CFU/μg pUC19. See "Transformation procedure for TransforMax™ EPI300™ Electrocompetent E. coli" on page 35 for transformation using electrocompetent cells.

Transformation using chemically competent cells

1. Pre-chill 15 mL disposable polypropylene culture tubes (17 x 100 mm, one tube for each transformation reaction).

2. Thaw chemically competent cells on ice for 5–15 minutes.

3. Add 40 µL of thawed, chemically competent cells to each cold tube.

4. Add 2 µL of the assembly reaction to each cold tube of competent cells. Mix by briefly stirring (do not pipet up and down).

5. Incubate the cells and DNA on ice for 30 minutes without mixing.

6. Heat shock the cell/DNA mixture in a 42°C water bath for 45 seconds.

7. Return tubes to ice for 2 minutes.

8. Add 950 µL room temperature recovery media to the cells in the culture tube.

9. Incubate the tubes with shaking at about 250 rpm for 1 hour at 37°C to allow the cells to recover.

10. While cells are shaking, pre-warm LB plates with appropriate antibiotics in an incubator for 30 minutes.

Note: Pre-warm two plates for each clone.

11. At the end of the 1 hour incubation, plate 150 µL of the transformants on the LB plate with antibiotics. Use glass beads to swirl and plate evenly. Label plates with the following information:

• Plate Name

• Plating volume (150 µL)

• Plating Date

• Initials

NOTE: If you are using competent cells other than 10G cells, plate all of the cells. You may need to centrifuge tubes for 1 minute and discard more than half of the clear supernatant in order to plate all of the cells.

12. Place the plates in a 37°C incubator upside down, overnight.

35

Transformation procedure for TransforMax™ EPI300™ Electrocompetent E. coliWe recommend diluting cloning reactions 1:2 with molecular biology grade water before transformation.

1. Add 1 mL SOC media to a 15-mL culture tube (one tube per reaction). Label tubes and place on ice for 10 minutes.

2. Chill clean electroporation cuvettes on ice.

3. Pipet 50 µL of EPI300™ cells (Lucigen Cat. No. EC300110) directly between the slit of the cuvettes on ice (one cuvette per reaction).

4. Add 2 µL of the assembly reaction to the cells in the cuvette. Mix by pipetting up and down gently two times. Place the cuvette back on ice.

5. Incubate cuvette on ice for one minute.

6. Gently tap cuvette on a benchtop two times to make sure all contents are at the bottom of the cuvette in between the slit.

7. Insert the cuvette into a Bio-Rad electroporator or equivalent, and press PULSE.

NOTE: Pulse Settings for EPI300™ cells are 1200 V, 25 uF, 200 Ω, 0.1 cm cuvette.

8. While the pulse is taking place (≈2 seconds), remove about 800 µL SOC from a pre-chilled 15 mL tube (step 1), and immediately add the SOC to the cuvette after the pulse.

9. Gently mix the cells and SOC by pipetting up and down 2–3 times. Add the cell and SOC mixture back into the tube containing the remaining 200 µL SOC.

NOTE: Work as quickly as possible until the cells are transferred into the 15 mL culture tube.

10. Incubate the cells and SOC for 1 hour at 37°C with shaking at 220–230 rpm.

11. While cells are shaking, pre-warm LB plates with appropriate antibiotics in an incubator for 30 minutes.

Note: Pre-warm two plates for each clone.

12. At the end of the 1-hour incubation, plate 50–100 µL of cells on LB plate with appropriate antibiotics.

NOTE: Use glass beads to swirl and plate evenly. Label plates with the following information:

• Plate Name

• Plating volume (50–100 µL)

• Plating Date

• Initials

13. Place the plates in a 37°C incubator upside down, overnight.

36

Full-length Clone (EFC) AnalysisScreening colonies

We typically pick 2–6 colonies and screen clones by colony PCR or by plasmid DNA purification followed by restriction enzyme digestion and sequencing. See the next sections for additional information.

If you are using a blue-white screening vector, such as pUCGA 1.0, refer to the following table for guidance.

Screening pUCGA 1.0 colonies

Colony color Indicates

White Successful cloning reaction (not necessarily error-free clones)

Blue Unsuccessful cloning reaction

Confirming full-length clones

The two primary methods for confirming full-length clones, double restriction enzyme digestion and PCR amplification, are discussed in the following sections.

Restriction enzyme digestion (Optional)Perform a double digestion using appropriate restriction enzymes. For the pUCGA 1.0 vector, use Xbal and BglII to release the BioXp™-synthesized fragment from the vector.

Note: Be certain that your fragments do not also contain Xbal and BglII recognition sites within the sequence.

PCR amplification (Optional)

1. For each clone, prepare the following reaction mixture in a thin-walled PCR tube:

Component Final concentration Volume

pDNA template ≤0.1 µg/µL χ μL

PCR Primer Mix (25 µM)*† 0.5 µM 1.0 μL

Q5® High-Fidelity 2X Master Mix (NEB, Catalog number M0492) †

1X 12.5 μL

Nuclease-free water † — to 25 μL

Total 25 μL

* Each primer is 12.5 µM† User provided

2. Transfer tubes to a thermocycler. Program and start the thermocycler using the following conditions:

Initial denaturation 98°C 1 minute 1 Cycle

Amplification 98°C 10−30 seconds

30 Cycles60°C 30 seconds

68–72°C 30–45 seconds

Final extension 72°C 5 minutes 1 Cycle


The following primers can be used to amplify the insert of pUCGA 1.0 clones:

Forward primer 5’: 5’-GAGCACATCTCGTTCGCTATTCAGGGATTG-3‘

Reverse Primer 3’: 5’-CATCCACGCCTGAGATCTAGACAAACTTCC-3‘

37

Sequencing

Sequence only full-length clones to identify error-free clones. Note that cloning efficiency and sequence fidelity are affected by:

• Chosen cloning method

• Chosen transformation method

• Clone length

• Complexity

• Interaction with the cell clone host (i.e. some sequences may be toxic to E. coli thereby causing host-induced mutations)

We recommend screening by colony PCR or restriction enzyme digest to identify full-length clones prior to sequencing. The number of full-length clones that need to be sequenced to identify an EFC (with 95% confidence) is as follows:

DNA fragment Size Number of Clones

< 1000 bp 2–3

1000 – 1500 bp 3–5

1501 – 1800 bp 4–6

Cloning efficiencyCloning efficiency is dependent on a number of factors including the size of the insert and complexity of the construct. The following figure shows the calculated cloning efficiency following transformation using pUCGA 1.0 clones obtained from 13 independent BioXp™ Cloning Module runs with DNA inserts between 400–1800 bp.

The high observed cloning efficiency from the BioXp™ 3200 System is shown in the following figure. As expected, highest cloning efficiencies are from smaller DNA fragments. DNA fragments were assembled and cloned on the BioXp™ 3200 System. Following transformation and an overnight incubation, colonies were counted and restriction enzyme-mapped. The total cloning efficiency (CE) for full-length inserts was calculated using the following formula:

CE (% )= (# of colonies containing full-length insert / total # of picked colonies) x 100

Small DNA fragments

Medium DNA fragments

Large DNA fragments

100

90

80

70

60

50

40

30

20

10

0

Full-

Leng

th C

loni

ng E

ci

ency

(%)

(430–890 bp) (1120–1416 bp) (1580–1810 bp)

n=94

n=79

n=68

Figure 6. High cloning efficiency from constructs generated on the BioXp 3200 System. After collecting constructs from the BioXp System deck following assembly and pUCGA 1.0 vector cloning runs using constructs with a range of fragment lengths and GC percentages, we performed transformation, plasmid preparation, and screening with double restriction enzyme digestion. The overall cloning efficiency of full-length pUCGA 1.0 clones is 83%. Additionally, we grouped clones according to fragment size for further cloning efficiency analysis. As expected, the highest cloning efficiency (> 90%) is observed for the smallest DNA fragments (< 900 bp).

38

Reference MaterialpUCGA 1.0 Amp clone mapA simplified clone map with sequence adjacent to the custom insert of a pUCGA 1.0 clone is shown below.

39

TroubleshootingIssue Possible Cause Inquiries and Recommendations

Absence or low amount of product

Challenging sequences or suboptimal protocol for sequences assembled

Review sequence complexities with Customer Service. Contact Technical Support.Note: De novo DNA synthesis is construct-dependent.

Suboptimal purification of BioXp™ Tiles during the job run

Using freshly prepared 70% ethanol is critical for successful DNA assembly.

Non-specific bands or faint or weak product bands

Challenging sequence dynamics may cause poor to low DNA synthesis

Gel purify the band of the correct size from an agarose gel and continue downstream applications.Note: De novo DNA synthesis is construct-dependent.

High molecular weight bands

Construct-dependent artifact High molecular weight bands may be concatemers and normally do not interfere with downstream applications such as cloning or amplification.Note: You may gel or column purify the final DNA assembly product to remove high molecular weight band(s).

No bands No final DNA assembly product is in the recovery plate

Using freshly prepared 70% ethanol is critical for successful DNA assembly.It is possible that the construct failed to synthesize due to complexity. Confirm that the construct meets the recommended specifications (Green sequences).Contact Technical Support for additional help.

Instrument error Incorrect loading of reagents on the instrument deck

Confirm proper loading of the instrument deck (see the reference image on page 27).

Low number of colony-forming units

Low E. coli transformation efficiency (TE)

Test the transformation efficiency of the cells. Be certain to use high transformation efficiency competent cells (>1 × 109 CFU/μg pUC19) such as Lucigen E. cloni® 10G cells.Dilute the Gibson Assembly® reaction 1:2 with molecular biology grade water before transformation.

Poorly assembled DNA Electrophorese the BioXp™ Tiles (from the Oligo Vault™ Plate) on a 1–2% agarose gel to analyze assembly reaction product.

Compromised final clone product

If electrophoresis of the BioXp™ Tiles is as expected, but the reaction volume of the final cloned product is less than 17 µL, an error may have occurred during the final cloning stages of the system run.

Improper plating Be certain to plate transformants on LB plates containing appropriate antibiotics.

Low cloning efficiency

Poorly assembled DNA Electrophorese BioXp™ Tiles (from the Oligo Vault™ Plate) on a 1–2% agarose gel to analyze assembly reaction product.

Improper plating Be certain to plate transformants on LB plates containing appropriate antibiotics.

Absence of an error-free clone

Sequence-specific cause Tandem or repetitive sequences may prove difficult to assemble and clone. Sequences with DNA complexity beyond the recommended specifications may prove difficult to assemble and clone.Host cell toxicity.

Insufficient number of colonies analyzed

We recommend picking 2–6 colonies for analysis (see page 37 for detailed recommendations). For inefficient assemblies with the presence of by-products, you may need to increase the number of colonies analyzed in order to identify error-free clones. Consider performing agarose gel electrophoresis of the BioXp™ Tiles, located in the Oligo Vault™ Plate, to determine the efficiency or success of the assembly reaction.

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