FALL 2009/SPRING 2010 PREP GUIDE BIO:219 BIOTECHNOLOGY Iusers.stlcc.edu/departments/fvbio/Bio219_Lab_Manual/… · · 2010-01-12prep guide bio:219 biotechnology i table of contents

EILENE LYONS – REVISED 1/12/2010 1

FALL 2009/SPRING 2010

PREP GUIDE

BIO:219 BIOTECHNOLOGY I

TABLE OF CONTENTS Setup for first day of class.....................Page 2

Lab 1 Genotyping of Arabidopsis.................Page 3-6

Lab 2 DNA Concentration and Purity .......... .....Page 7-8

Lab 3 PAGE.......................................Page 9- 10

Lab 4 Purification of Eco RI(EDVOTEK Kit 302)....Page 11-12

Lab 5 Mammalian Cell Culture...................Page 13 - 14

Lab 6 Pop Bead Cloning (dry lab)....................Page 15

Lab 7 DNA Restriction for Cloning...................Page 16

Lab 8 Gene Clean....................................Page 17

Lab 9 Ligation.................................... Page 18

Lab 10 Transformation & Blue-White Selection ....Page 19-20

Lab 11 Plasmid DNA Isolation (Miniprep) ........... Page 21

Lab 12 Recombinant DNA Restriction and Mapping .....Page 22

Lab 13 Manual DNA Sequencing (EDVOTEK Kit 341)...Page 23-25

Lab 14 Gene Expression (IL-8 ELISA KIT; Qiagen RNA cleanup

kit)............ ........... ........... .......Page 26

*Items marked with an asterisk do not need to be place on the

cart, but should be available in the room or in the immediate prep

area.

BIOTECHNOLOGY I – FIRST MEETING OF THE SEMESTER


BIOTECHNOLOGY I FIRST DAY SETUP


FIRST DAY SETUP Clean lab coats – all sizes

Colored water for pipetter practice

MATERIALS for growing Arabidopsis – see Lab 1 prep sheet

FOR EACH TEAM – set up one bin for each team, please

automatic micropipetters - one each size personal microcentrifuge & both rotors

beaker labeled for used tips plastic dish for staining/holding gels

Kim wipes scissors

loading dye - 6x or 10x Sharpie Markers -2

mm ruler sterile tips -one box of each kind of

microcentrifuge tubes - 1.5 ml -sterile tube rack -15 ml

molecular grade water - 10 mL tube rack - 1.5 ml

IN THE CLASSROOM PERMANENTLY

Please make sure there is container for dirty glassware during each lab

Agarose – electrophoresis grade

Buffers: 50x TAE, 50x TBE, 1x TNE buffer, 10:1 TE buffer

Carboy of dH2O

Colored Owl or Fisher horizontal gel rigs with combs

Conical tubes, Fisher, 15 ml and 30 ml and racks

Coverage – one bottle per table

Cuvettes, cuvette racks

Electronic balances, weigh boats, spatulas and paint brush for clean up

Ethidium Bromide [10 mg/ml] stock (keep refrigerated – small refrigerator?)

Ethidium Bromide disposal station (for disposal of buffer and gels)

Glassware, including beakers, 250 ml flasks, graduated cylinders, Corning bottles

Heating block with holes for 1.5 ml tubes - 2 Kim wipes

Lab diapers

Labeling tape

Microcentrifuges – Eppendorf type – at least 2

Microcentrifuge tubes, 1.5 ml and racks

Microwave oven and hot gloves

Parafilm

Plastic wrap

Serological pipette pumps – manual and electric

Serological pipettes – all sizes

Sterile tips – all sizes

Transfer pipettes

Water bath and floating tube holders

Vortex mixers

Zip Lock bags for storing gels

ITEMS IN THE PREP AREA WHERE STUDENTS CAN ACCESS EASILY:

Ice buckets – leave in the ice room by ice machine

Power supplies

Rockers and shakers

UV spectrophotometers – 2

UV transilluminator, camera, film, face shields, spatulas

BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR


LAB 1 GENOTYPING OF Arabidopsis TIMELINE

Day 1: Plant Arabidopsis seeds and prepare solutions

Day 2: DNA extraction and PCR

Day 3: Gel electrophoresis and analysis

MATERIALS for growing Arabidopsis Arabidopsis seeds (Carolina Biological Supply or [email protected] for seed ordering)

Each student needs about 25-50 clf-2 seed (from a heterozygous plant);

Modified MS agar plates, one per student

Growth chamber set on continuous light

Bleach for 30% bleach solution (students will prepare)

Sterile dH2O – one tube or bottle per group

Sterile 0.1% agarose solution (students will prepare)

Aluminum foil

MATERIALS for DNA Isolation and PCR

2 per group Ready-To-Go PCR Beads (PuReTaq Ready-To-Go™ PCR Beads from

http://www1.amershambiosciences.com 96 reactions #27-9559-01, NC9711585 from Fisher, will

last ≥ 1.5 years; cost $227.50)

Each pellet reconstituted to 25 μL contains 1.5 units Taq polymerase, 10 mM Tris-HCl

(pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 200 μM of each dNTP.

Primers can be ordered from http://invitrogen.com

0.4 uM minimum CLF (wildtype) primers/loading dye mix – 25 μL per Team

FORWARD 5’-TTAACCCGGACCCGCATTTGTTTCGG-3’

REVERSE 5’-AGAGAAGCTCAAACAAGCCATCGA-3’

0.4 uM minimum clf-2 (mutant) primers/loading dye mix – 25 μL per Team

FORWARD 5’-TTAACCCGGACCCGCATTTGTTTCGG-3’

Ds REVERSE 5’-GTCGGCGTGCGGCTGGCGGCG-3’

Edward’s Buffer, 2 ml per team (Students will prepare)

Cresol Red Loading Dye

Isopropanol, aliquoted – 2 ml per team

Pellet pestles – one per team

Dissecting microscopes – 1 per team

Thermal cycler

Hair dryer

TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) – 500 μl per team

MATERIALS for Electrophoresis

100 - 1000 bp ladder or pBR322 DNA-BstN I digest (inexpensive marker from NE Biolabs)

*Heating block or water bath set at 50°C

*50X TAE electrophoresis buffer (students will dilute to 1X working solution)

*D.C. power supplies – per 2 groups

*Practice loading dye

*[10 mg/ml] ethidium bromide stock solution

*Should be in room – do not place on cart


EILENE LYONS – REVISED 1/12/2010

5

5

RECIPES:

Modified MS agar:

4.3 g MS salts (half the amount used for bacteria culture)

900 ml distilled water

Use 1.0 M NaOH or KOH to adjust to pH 5.7

BTV of 1 Liter

Then add 8 g agar and autoclave for 20 minutes at 120°C. One liter makes 40-50 10 cm

diameter plates.

Cresol Red loading dye

1% Cresol Red Dye Stock – yield 50 ml

Add 500 mg to 50 ml of ddH2O in a 50 ml tube or bottle

Shake to dissolve

Store at room temperature

Cresol Red Loading Dye – yield 50 ml of working solution

Add 17 g of sucrose to 49 ml of ddH2O in a 50 ml tube or bottle

Shake to dissolve

Add 1 ml of 1% Cresol Red Dye

Shake tube to mix. Store at 4°C.

Edward’s Buffer – yield 50 ml (solid NaCl and concentrated stocks will be used)

Mix the ingredients in a 50 ml bottle (can be stored at room temperature, indefinitely).

32.5 ml ddH2O

10 ml of 1 M Tris pH 8

2.5 ml of 5 M NaCl

2.5 ml of 0.5 M EDTA

2.5 ml of 10% SDS

50x Concentrated TAE Electrophoresis Buffer (40 mM Tris-acetate, 2 mM EDTA)

Add the following to dH2O to give a final volume of 1 liter

242 g Tris base (Tris [hydroxymethyl] aminomethane)

57.1 ml glacial acetic acid

100 ml 0.5 M EDTA (pH 8)

Students will dilute to a 1X working solution

Primer/Loading Dye Mix

Final concentration of components:

0.25 picomoles/μL of each primer, 13.9% sucrose, and 0.0082% cresol red in Tris-low

EDTA (TLE) buffer (10 mM Tric-HCl, pH 8.0; 0.1 mM EDTA).



6

6

The ideal concentration is between 0.1 and 0.5 M. Primers are sold as solid DNA by

some companies and diluted in water by other companies. A certificate of analysis

packed with the primers give the mass, number of moles and if diluted, the concentration

in either μM or μg/μl or both. It can be difficult to determine how to dilute and mix the

primers for use in PCR so that the concentration is between the optimum 0.1 and 0.5 μM.

Table 1 and the sample calculation, below, may help.

Table 1. Molar conversion for Primer Concentration* Primer length pmol/µg 20 pmol** 18-mer 168 119 ng

20-mer 152 132 ng

25-mer 121 165 ng

30-mer 101 198 ng

* From Qiagen News, Issue 5 1997

** 20 pmol of primer in a 100 µl PCR reaction gives a primer concentration of 0.2 µM

SAMPLE CALCULATION for DILUTION OF LIQUID PRIMER DNA Suppose a primer concentration is given as 40 μM and 0.32 μg/μl. You need to make up 300 μl of primer/loading dye mix. The protocol states that 22.5 μl of the primer/loading dye is used in the 30 μl reaction. The final concentration of each of the primers in the 30 μl reaction must be 0.1 – 0.5 μM. 1. The primer’s final concentration in the reaction must be 0.1 – 0.5 μM, so dilute the

primer to 0.5 μM and use this to set up the reaction. 2. Use C1 x V1 = C2 x V2 to calculate the volume of working solution to use.

C1 = 40 μM

V1 = X C2 = 0.5 μM V2 = 300 μl

(40 μM) X = (0.5 μM) (300 μl) X = 3.75 μl

Add 3.75 μl of the primer as supplied by the manufacturer to enough loading dye and the other reagents to give a total of 300 μl. When 22.5 ul of this mix is used in the reaction, the final concentration of this primer will be 0.375 μM, which is within the 0.1 – 0.5 μM final concentration limits.

(0.5 μM) (22.5 μl) = X (30 μl) X = 0.375 μM



7

7

SAMPLE CALCULATION for DILUTION OF DRY PRIMER DNA Suppose a primer is 280 μg and 50 nmoles and you need to make up 300 μl of primer/loading dye mix that has a final concentration of 0.25 picomoles/μl.

1. Dilute the primer to make a 100 pmole/μl stock solution. Add 500 μl of dH20 to dissolve the DNA.

50 nmoles = 50,000 pmoles 50,000 pmoles/500 μl = 100 pmoles/μl

2. Dilute 1 μl in 9 μl dH20 to give a working solution of 10 pmoles/μl. 3. Use C1 x V1 = C2 x V2 to calculate the volume of working solution to use. C1 = 10 pmoles/μl

V1 = X C2 = 0.25 pmoles/μl V2 = 300 μl

(10 pmoles/μl) X = (0.25 pmoles/μl) (300 μl) X = 7.5 μl

4. Add 7.5 μl working primer solution to make the primer/loading dye mixture.

BIOTECHNOLOGY I LAB 2 DNA CONCENTRATION AND PURITY


LAB 2 - DNA CONCENTRATION AND PURITY

ITEMS TO ORDER IMMEDIATELY Commercial Lambda DNA (0.3-0.5 μg/μL)

DNase

High Mass Molecular Weight Marker DNA

TIMELINE

This lab will take 1 laboratory period

MATERIALS for PART I. Prep

10X TNE Buffer (please check to make sure there is no contamination in the bottle;

buffer may need to be filtered)

MATERIALS for PART II. 0.8% Gel Casting *Powdered agarose

*50x Concentrated TAE Electrophoresis Buffer (Students will dilute to 1X)

*Horizontal gel electrophoresis boxes – one/team

*Electronic balance, weigh-boats and spatulas

*Microwave oven and hot gloves

*Electronic pipette pumps

*Thumb-operated pipette pumps

*Serological pipettes – 10 ml

*100 ml graduated cylinder - one/team

*250 ml flasks - one/team

*Dishpan in sink for dirty glassware

*Scissors – one/team

*Sharpie marking pens

MATERIALS for PARTS III & IV Electrophoresis and UV Spectrophotometry

Commercial Lambda DNA (0.3-0.5 μg/μL) Place this and other DNA on ice for lab

1/20 dilution uncut concentrated Lambda DNA to use as unknown DNA samples – 50 μL

in a 1.5 mL tube labeled as DNA A per team – do not write concentration on tubes

1/10 dilution uncut concentrated Lambda DNA mixed 1:1 with Bovine Serum Albumin

to use as DNA B – 50 μL in a 1.5 ml tube per team

Degraded DNA – 50 μL in a 1.5 mL tube labeled as DNA C per team (add DNase)

REPORT DNA SAMPLE CONCENTRATIONS TO THE INSTRUCTOR

Molecular weight DNA ladder – one 1.5 ml tube per team

*6x or 10x Gel Loading Solution – one 1.5 ml tube per team

*Practice Gel Loading Solution – one 1.5 ml tube per team

*50X TAE electrophoresis buffer (students will dilute to 1X)

*1x TNE Buffer – two 50 mL bottles

*2 Beckman UV Spectrophotometers and matching quartz cuvettes for each

*Kim wipes

*Ethidium bromide [10 mg/ml]

* Place on cart if not in the storage cabinets in the lab

BIOTECHNOLOGY I LAB 2 DNA CONCENTRATION AND PURITY


9

9

RECIPES for Lab 2

10x TNE Buffer stock solution:

100 mM Tris base (Tris [hydroxymethyl] aminomethane)

10 mM EDTA

2.0 M NaCl

Adjust pH to 7.4 using concentrated HCl and a pH meter. Dilute to 1x working

concentration using dH2O. Filter sterilize to remove all precipitate and contaminants.

50x Concentrated TAE Electrophoresis Buffer (40 mM Tris-acetate, 2 mM EDTA)

Add the following to dH2O to give a final volume of 1 liter

242 g Tris base

57.1 ml glacial acetic acid

100 ml 0.5 M EDTA (pH 8)

BIOTECHNOLOGY I LAB 3 PAGE


LAB 3 POLYACRYLAMIDE GEL ELECTROPHORESIS OF PROTEINS

TIMELINE

DAY 1: Prep for the lab, extract plant proteins, run SDS PAGE, stain and destain

DAY 2: Graph results using MS Excel and analyze

MATERIALS:

Per class

Bio-Safe Coomassie Blue stain (destains with dH2O)

*Practice gel loading solution

10X Tris-glycine-SDS buffer stock

*Vortex mixers

*Heating block set at 95°C

*1000 mL graduated cylinder

*dH2O

*1 L Corning orange capped bottles

*Plastic wrap

White light box

*Polaroid camera and film

*A supply of transfer pipettes

*Labeling tape

*1.5 ml microfuge tubes

Per every 2 teams

Flat metal spatula for separating gels

Dual vertical mini gel rig with clamps – please put on cart

Power supply – please put on cart

Per team

Kaleidoscope prestained protein molecular weight markers – aliquot 25 μl per team (Bio-

Rad # 161-0324) NOTE: heat briefly at 37°C to dissolve any precipitated SDS before aliquoting.

Flowering plants, one/team

2x Protein loading dye/buffer (≥ 100 ul)

Scalpel with sharp blade

Precast 15% polyacrylamide gel for SDS PAGE

8 disposable pellet pestles

2 ml 1X Laemmli buffer


BIOTECHNOLOGY I LAB 3 PAGE


11

11

RECIPES

10x Tris-Glycine-SDS Buffer (500 ml should be allowed for each double gel rig; 1x =

25 mM Tris; 192 mM glycine, 0.1 % SDS )

3.04 g Tris base

14.41 g glycine

1.0 g SDS

dH2O to give 100 ml final volume

Dilute 100ml in 900ml dH2O for 1x working concentration

10 x Laemmli Buffer

0.25 M Tris,

1.92 M Glycine

1 % SDS in aqueous solution

Dilute to 1x working concentration

2x Protein loading dye (10 ml)

1.2 ml 1M Tris HCl pH 8**

4 ml 10% SDS

2 ml 100% glycerol

1 mg bromophenol blue

0.1 ml -mercaptoethanol

2.7 ml dH2O to give 10 ml final volume

**Adjust to pH 8 with HCl for prepoured graduated gels; if using discontinuous self poured

gels, adjust pH to 6.8.

BIOTECHNOLOGY I LAB 4 PURIFICATION OF ECO RI BY ION EXCHANGE


LAB 4

PURIFICATION OF ECOR I by ION EXCHANGE CHROMATOGRAPHY

Edvotek Kit 302 is used for this lab

TIMELINE Day 1 – Prep, collect column fractions, cast agarose gels

Day 2 – First Assay

Day 3 – Second Assay

MATERIALS & EQUIPMENT – DAY 1 Chromatography columns, one per group

Ring stand with clamps, one per group

Ten 13 x 100 mm test tubes per group

(C) 10x Equilibration Buffer

Prep personnel should prepare the following

(B) DEAE-Cellulose (Please prepare 30 minutes before class per instructions, below)

(A) E. coli RY extract, lyophilized – must be re-hydrated per directions below

Equilibration Buffer working solution – DO NOT PREPARE UNTIL 10X HAS BEEN

USED TO HYDRATE THE DEAE-CELLULOSE

KCl buffers [0.1M], [0.2M], [0.5M] – each group will need 6 mL of each

Students will aliquot the following from the kit – Please place on the cart

(F) Eco RI Reaction Buffer

(H) Lambda DNA – 0.2 μg/μl concentration

(I) Lambda/Eco RI Marker

(J) Eco RI Dilution Buffer

Check the room/prep shelves for availability of the following: *50x TAE Electrophoresis Buffer (also in kit)

*15 ml conical centrifuge tubes

*Agarose powder

*Gel electrophoresis units, one per group

*5 ml serological pipette and pump, one per group

*Microwave oven & hot gloves

*Balances, spatula, weigh boats, cleaning brush

*Ice buckets

*1 Liter Corning bottles with lids

*[10 mg/ml] Ethidium bromide solution

*Should be in room or students can get – do not place on cart

RECIPES AND DIRECTIONS FOR DAY 1 PREP

DEAE-Cellulose Matrix

Add 35 ml of 10x equilibration buffer (C) to the bottle of DEAE-Cellulose (B). Cap

tightly and place on a rocker or orbital shaker to hydrate for at least 30 minutes. Aliquot 6

ml for each of the six groups. (The Instructor may have to do this)

(Recipes continued on the next page)

BIOTECHNOLOGY I LAB 4 PURIFICATION OF ECO RI BY ION EXCHANGE


DAY 1 PREP (CONT.)

Equilibration Buffer Working Solution

Mix the following and stir thoroughly:

350 ml dH2O

50 ml 10x Equilibration buffer (C)

100 ml 50% glycerol (D)

KCl Buffers

0.1 M KCl: use 0.75 g KCl and BTV of 100 ml with Equilibration Buffer working

solution


solution


solution

E. coli Cell Extract containing Eco RI restriction enzyme

1. Re-hydrate the sample by adding 0.5 ml of ddH2O to tube component A and let sit for

5 minutes.

2. Mix by vortexing on high speed and transfer the entire contents to a 50 ml conical

tube. Rinse tube A six times – each time with 1 ml of Equilibration Buffer working

solution and add the rinse material to the 50 ml conical tube. Mix the contents well.

3. Label a tube for each of 6 lab groups as “Cell Extract” and add 1 ml of the re-

hydrated extract to each tube. Store this extract on ice.

MATERIALS AND EQUIPMENT - DAY 2 and DAY 3 *37°C Waterbath and floating tube holders (Each group will have 9 reaction tubes)

*65°C heating block with holes for 1.5 ml tubes (Each group will have 9 reaction tubes)

Lambda DNA diluted to 4 ng/uL; aliquot 25 ul for each group (this is needed for the gel

for the second assay)

[10 mg/ml] Ethidium bromide stock

*2 UV Spectrophotometers

*UV or Matched Quartz Cuvettes

*Dishes for transporting gels

*Plastic wrap

E-gels and E-gel rigs – one per team


BIOTECHNOLOGY I MAMMALIAN CELL CULTURE


LAB 5 – MAMMALIAN CELL CULTURE Here are a couple important things to prep before bringing up cells:

1. Sterilize the carbon dioxide incubator (for the older CO2 incubator, clean with 70% EtOH and

wipe with Beta-iodine, including shelves. If necessary, add water in the bottom after cleaning

and add Fisher Clear Bath.)

2. PLEASE CLEAN THE WATERBATH that will be used to warm culture media - wash with

antibacterial soap or detergent and add Clear Bath.

3. Autoclave media bottles- 100ml bottles. This is important to ensure no contamination. Even if

they have been already autoclaved once, couple days before autoclave again to ensure sterile,

remind whoever is doing this not to touch near the neck or bottle top. Store these bottles away

from any bacteria plates or incubators.

4. Pay special attention not the leave any supplies that will be used for the lab near bacterial

plates, incubators, or anything of the sort.

5. If instructor is NOT having students bring up cells, bring them up and passed at least once, to

make sure they are healthy. Do this the week prior to the lab. ATCC instructions come with

the cells.

TIMELINE: This lab will be continued through the remainder of the semester

MATERIALS

TO ORDER:

Cell scrapers (check with instructor to determine if she will use these or Trypsin-EDTA)

Please make sure all HEPA filters on the incubator and the safety cabinets are fresh

CO2 gas

NIH 3T3 Cells from ATCC (mouse embyonic cell line), item CRL-1658 ($203 Betsy’s price)

Then to grow the cells: (all reagents from Sigma)

D5796 media 6 x 500 mL $87.10

N4637 serum 500 mL $48.40

T3924 trypsin 2 x 100 mL $8.70 each

H6648 salt 6 x 500 mL $73.30 (this is Hank's balanced salt solution, if you want to

just have PBS made up, that is fine too)

PREPARE:

100X penicillin/streptomycin (check with instructor before ordering – may not be used)

Trypan Blue – aliquot 50 µL per 1.5 ml microcentrifuge tube per team

0.05% Trypsin-EDTA

Phenol red (pH 6.8-yellow – 8.2-red indicator)

Sterile Phosphate Buffered Saline (PBS), 1M OR Hank’s balanced salt solution

Sterilize CO2 Incubator

For Bringing up Cells prior to student lab

Flasks- T-25, T-75

Media: 100 ml of 10% FBS DMEM (+ Antibiotics, if instructor wants this) – students may

prepare media themselves

*Serological Pipettes

*15 ml Conical Centrifuge Tubes (Sterile for Serum and Antibiotics)

*sterile 50 ml tubes and Autoclaved or Sterile 100 ml Media bottles (Glass is better)

BIOTECHNOLOGY I MAMMALIAN CELL CULTURE


Per Class (Amounts depend on # of teams):

*Confluent cell cultures in T-25 flasks, started 1 week prior

*DMEM or other media (in cold room)

10% Fetal Bovine Serum (FBS)

Sterile Phosphate Buffered Saline (PBS), 1M or Hank’s – divided into two bottles

Autoclaved or Sterile 100 ml Media bottles (Glass is better)

*5 ml, 10 ml, 25 ml serological pipettes

Cell scrapers (if using)

Flasks - T-25

Spray bottle of 70% EtOH

*Parafilm

*15ml tubes and holders (racks)

Hemocytometers – one per team

Cell counters one per team

Trypan Blue –50 µL per team

1 100ml Media Bottle per team

*sterile 50 ml tubes

*Light microscopes

Inverted light microscope – 1 with camera and computer and another 1 or 2 without

Water bath (clean the one in the lab and replace with fresh water)

*Labeling tape

To be left in the BSCs in the prep area and micro lab

10% bleach solution

container for used pipettes

two electric pipette pumps

beaker for old media sterilization


BIOTECHNOLOGY I LAB 6 POP BEAD CLONING


LAB 6

POP BEAD CLONING

DNA model

Pop bead cloning kit – please check to make sure each bag contains the correct number of

beads.

MATERIALS In your bead kit, you should have one linear set of beads in the following order:

Hole end, 13 White, 2 pink, 1 white, 1 pink, 1 yellow, 2 white, 2 red, 2 yellow, 4 red, 2

white, 2 yellow, 2 white, 1 pink, 1 yellow, 7 white, knob end.

There should also be one circular set of beads in the following order, starting after the

twisted white bead:

Hole end, 5 orange, 2 pink, 1 orange, 3 blue, 1 pink, 1 yellow, 3 blue, 1 orange, 2 yellow,

2 orange, 1 twisted white bead- knob end.

BIOTECHNOLOGY I LAB 7- DNA RESTRICTION FOR CLONING


LAB 7 DNA RESTRICTION FOR CLONING

NOTE: Each team will set up one set of two digestions (pUC and pAMY).

TIMELINE: Students will set up digestions and run the results on an E-gel to confirm,

all in one class period

MATERIALS - DNA Digestion

Stock tube of pUC 18 DNA approx. [0.5 ug/ul]; students will dilute per instructor’s

directions

*Eco RI endonuclease

*10x buffer for Eco RI, 50 µL/team

*molecular grade dH2O

*Water bath set at 37 C

*automatic micropipetters and tips

*used tip container

*Kim wipes

*1.5 ml microcentrifuge tubes

*microcentrifuge tube rack

*Sharpie marking pen

Floating microfuge tube racks for water bath

MATERIALS - Gel Electrophoresis

E-gels and rigs

Uncut pUC 18 DNA as control on gel

Molecular Weight Marker DNA (with 10 L concentration labeled)

*dH2O


*Microfuge tube rack

*UV Transilluminator

*UV Camera and film

*UV eye protection and face shields


BIOTECHNOLOGY I LAB 8 GENE CLEAN


LAB 8

Gene Clean

TIME LINE:

Day 1: Cast agarose gels for gene clean (on day of last lab.)

Day 2: Run the gel and extract the DNA fragments and gene clean; run the gene clean

product on an E-Gel to verify and quantify Mrs. Lyons will start the gel 2 hours before

class starts.

MATERIALS

55 C & 65°C water baths (with floating tube racks) – please make sure there are two in the lab

Please make sure 2 or more Eppendorf microcentrifuges are in the lab.

Sigma GenElute Gel Extraction Kit – Please dilute enough of each solution so that each

student has a 1.5 ml tube of each for his/her own gene clean.

*1X TAE buffer

Sterile swabs

*Student DNA digestions

Molecular Weight Marker DNA

*Kim wipes

*D.C. power supply


*UV Camera and film

*UV safety goggles and face shields

*[10 mg/ml] Ethidium Bromide stock

*6x or 10x gel loading dye

single-edge razor blades or scalpels

An E-gel and rig for gene clean

verification

*Molecular grade water

*disposal for Ethidium Bromide waste


BIOTECHNOLOGY 1 LIGATION


LAB 9

LIGATION

MATERIALS

per class

*T4 ligase (check to make sure we have and let instructor know where it is)

*Gene cleaned insert DNA

*Digested plasmid DNA

Cooling block set at 16 C

*Vortex mixers – one per each end of lab bench

*Labeling tape

per Team

*Molecular grade or qualified water

*1.5 microcentrifuge tubes

10X Ligase buffer

ATP - 20 mM

*ice and ice bucket

*personal microcentrifuge


BIOTECHNOLOGY I TRANSFORMATION


LAB 10

TRANSFORMATION Order 95 mm x 15 mm plates – we will try larger plates this year.

Note: Each team will plate transformed culture onto 3 large plates. Two teams will also plate

negative control transformations onto 3 plates each and three teams will plate 3 positive control

plates each. Streak plates will also be made the day after and colonies picked off of that plate for

the miniprep, which means we need one more plate per team. So, 15 large plates are required

for the controls, plus 4 large plates per team, but have some extra, also. Make them all L-agar

+ Amp50

+ IPTG + X-gal.

MATERIALS For TRANSFORMATION

Per Class A tube of plasmid DNA from a previous semester for a control transformation (and back-up)

L-agar + Amp50

+ IPTG + X-gal plates – see above (room temp.)

JM109 Competent cells – 1 tube per team, and one for each of 5 controls

Recovery broth (SOC)

*Labeling tape to attach tubes to floor of shaker-incubator

*42 C heating block for transformation (check instructions with competent cells)

Shaker-incubator set at 37 C

Incubator set at 37 C for plates

Per team

*Qualified water

*1.5 ml microcentrifuge tubes – sterile – please check supply in room!

*ice and ice buckets with the following tubes to be assembled by the teams:

1 tube of supercoiled control DNA (supplied with competent cells)

-Mercaptoethanol (if supplied with competent cells – usually not with JM109)

Ligation reaction from previous lab

Qualified water

Sterile plastic spreaders for plating cells

*Personal microcentrifuge

MATERIALS FOR LIQUID CULTURE INOCULATION and GROWTH

Per class

Sterile toothpicks or sterile inoculating loops

Two bottles of 100 ml sterile L-broth media

Two 1.5 ml tubes of Ampicillin stock [10 mg/ml], thawed

10 ml (16 mm) sterile test tubes with loose fitting caps (two per student)

One test tube rack must be wired into the shaker incubator at an angle, so that the liquid cultures

shake at a slant and receive aeration, which is required for growth.

*Test tube racks – one more than the number of groups in the class

*Sterile 5 ml serological pipettes and pipette pumps

Shaker-incubator set at 37 C


BIOTECHNOLOGY I TRANSFORMATION


SOLUTIONS AND MEDIA for TRANSFORMATION

L agar-Amp50 -X-gal - IPTG plates (1 liter) Make plates within one week of the lab.

Make enough L agar for 40-45 mls per large Petri plate. Mix 10 g Bacto-tryptone (Difco

#0123-17-3), 5 g Bacto-yeast extract (Difco #0127-17-9), 10 g NaCl, 15 g Bacto agar (Difco #

0140-01), and cold ddH2O to make 1 liter. Use containers at least twice as large as the volume

of agar, or it may boil over in the autoclave. It is not necessary to boil the solution, as the

autoclaving will dissolve and mix the agar with the ddH2O. Autoclave 15 - 20 minutes at 15

p.s.i.

After agar has cooled to 50 C (agar is cool enough when you can hold your hand on the flask

with no discomfort), add sterile antibiotic (50 g/ml final concentration for ampicillin; use 10 ml

of 10 mg/ml sterile chloramphenicol). Final concentration of X-gal should be > 40 g/ml (use

3 ml 20 mg/ml sterile stock). Final concentration of IPTG should be > 120 g/ml (use 10 ml 20

mg/ml sterile stock). Stock solution instructions are below. (This is revised per Ginny after 04-

05.)

Pour into petri dishes. Let plates cool to room temperature. You may want to let them sit out

overnight at room temperature to get rid of the condensation on the lids. Sleeve plates, upside

down, in the plastic bag they came in, and store at 4 C. Bring to room temperature before using.

X-gal 20 mg/ml stock.

Dissolve 200 mg in 10 ml dimethyl formamide in a glass, not plastic, test tube with lid. Do not

filter sterilize. Store in dark (wrap aluminum foil around tube) at -20 C. Dimethyl formamide is

sterile.

IPTG 20 mg/ml stock

Dissolve 200 mg in 10 ml dH2O. Filter sterilize into sterile 15 ml centrifuge tube using a 0.22

micron filter attached to a 10 cc syringe.

Antibiotic stock solution- [10mg/ml]

Dissolve 100 mg in 10 ml ddH2O (or 95% ethanol for chloramphenicol). Filter sterilize into 10

sterile 1.5 ml microcentrifuge tubes using a 0.22 micron filter attached to a 10 cc syringe.

L-Broth (1 liter)

Use 10 g Bacto-tryptone (Difco #0123-17-3), 5 g Bacto-yeast extract (Difco #0127-17-9), 10 g

NaCl, and cold distilled ddH2O to make 1 liter. Aliquot to bottles. Autoclave 15 - 20 minutes at

15 p.s.i. After cooling, add sterile ampicillin (final concentration = 50 g/ml), if required.

SOC Broth

Buy pre-made. Contains the following:

2% Tryptone

0.5% Yeast Extract

10 mM NaCl

2.5 mM KCl

10 mM MgCl2

10 mM MgSO4

20 mM glucose

BIOTECHNOLOGY I MINIPREP


LAB 11

PLASMID DNA MINIPREP

MATERIALS

Miniprep

*Tubes of E. coli cultures grown overnight in shaking incubator

*Ice in ice bucket

*Kim wipes

*2 Eppendorf microcentrifuges we will not be using the personal microcentrifuges

*microfuge tube racks


Promega Wizard Miniprep DNA isolation kit components - Please aliquot enough of each

solution so that every student can do two minipreps

Mini spin columns

3 mL syringes and plungers


*container to dispose of bacterial waste

orange biohazard waste bag placed in the lab

*Coverage for cleaning bench tops

*10% bleach solution for disinfecting waste before disposal

Gel Electrophoresis

*plasmid DNA for + control on gel (instructor will get this)

*molecular grade water

E-Gels and rigs – one per every 2 teams (instructor should have E-gels)

Molecular Weight marker DNA

*Kim wipes


*UV Camera and film


* carboy of dH2O


BIOTECHNOLOGY I PLASMID MAPPING

Eilene Lyons Revised 1/12/10 23

LAB 12

RESTRICTION MAPPING TIMELINE

Day 1: Set up digestions of the recombinant plasmids that were constructed and isolated

in the previous labs; cast 0.7% agarose gels

Day 2: Run digestions on the gel, analyze results and construct the plasmid map(s).

MATERIALS:

PLASMID DNA RESTRICTION – DAY 1

per class

*Water bath set at 37 C with floatees

*Ice buckets with ice

*Thawed recombinant plasmid DNA


*Nalgene ice tray for enzymes

*Eco RI endonuclease

*Xba I endonuclease

*Hind III endonuclease

*Restriction enzyme reaction buffers H,

B, C, D and E

*Universal buffer for double digestion

*1.5 ml microcentrifuge tubes

*microcentrifuge tube racks

*personal microcentrifuges

*Sharpie permanent markers

*TE buffer (to be used for dilution of

uncut plasmid DNA)

GEL CASTING – DAY 1

Per class

*balance, spatula and weigh boats

*microwave oven & hot gloves

*250 ml flask per group

* carboy of dH2O

*agarose

*horizontal gel electrophoresis rigs

GEL ELECTROPHORESIS OF DIGESTED PLASMID DNA – DAY 2

Molecular weight marker DNA (NOT High mass ladder – 0.5 and 0.1 bp markers are required)

*metric rulers

PUC18 vector DNA


*used tip containers

*Kim wipes

*microfuge tube racks


D.C. power supply – one per 2 groups


*UV Camera and film


*50x TAE electrophoresis buffer

(students can dilute)

*10x gel loading solution


BIOTECHNOLOGY I DNA SEQUENCING


LAB 13

AUTOMATED DNA SEQUENCING MATERIALS

Heating block for 1.5 mL tubes, set to 95 C.

Molecular Grade (m.g.) sterile dH2O

m.g. 95% (v/v) ethanol/dH2O

m.g. 70% (v/v) ethanol/dH2O

3M Sodium Acetate pH5.2 – Sigma Cat. # S 7899

100 mM Na2EDTA pH8.0, prepared from 0.5 M Na2EDTA – Sigma Cat. # E 7889)

0.5 mL sterile microfuge tubes

Automatic micropipetters and tips

Thermal cycler with hot bonnet

Personal microcentrifuges with 0.5 tube rotor - one per group

Stop Solution (recipe below)

Materials from Beckman Coulter:

CEQ Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (No. 608120) which

contains:

Quick Start Mix

dNTPs

ddNTPs

Tris-HCl, MgCl2, reaction buffer – pH 8.9

Thermo Sequencase DNA Polymerase

Pyrophosphatase

pUC18 Control Template (0.25 g/ L)

M13 –47 Sequencing Primer (1.6 pmol/ L or 1.6 M)

Glycogen (20 mg/mL)

Mineral Oil

Sample Loading Solution (SLS)

Store the CEQ Cycle Sequencing kit at -20 C (frosty freezer, not frost free)

Recipes

Stop Solution (1.5 M NaOAc + 50 mM EDTA made fresh daily)

Mix equal volumes of 3M NaOAc and 100 mM EDTA

BIOTECHNOLOGY I ELISA


LAB 14

ELISA

MATERIALS

Per class:

Water bath set at 37 C

EDVOTEK Kit #271

HIV antigens (simulated – component A)

Positive control (1 antibodies – component B)

Donor 1 serum (simulated – component C)

Donor 2 serum (simulated – component D)

Anti-IgG-peroxidase conjugate (2 antibody – component E)

Hydrogen peroxide (component F)

Amiosalicylic acid (peroxide co-substrate – component G)

Phosphate buffered saline concentrate (component H)

Microtiter plates

Microcentrifuge tubes – 1.5 ml

Two 50 ml plastic tubes for mixing 2 antibody and substrate Scissors

Sharpie markers

Colored pencils

Tubes or bottles to hold 25 ml – one per student up to 10 per class (for PBS)

Beakers for waste solutions – one per two students

1 Corning bottle to hold 270 ml

Graduated cylinder to measure 270 ml

Electric serological pipette pumps – one per two students

1 ml serological pipettes – one per student

1 25 ml serological pipette

Bottles of dH2O – one per two students

BIOTECHNOLOGY I GENE EXPRESSION


LAB 15 GENE EXPRESSION IN MAMMALIAN CELLS

MATERIALS

Antibiotic media

10 ml Penicillin-Streptomycin

1 Liter F12 K media

110 mL Fetal Bovine Serum (FBS)

Filter sterilize

Trypsin-EDTA (1x)

1x Phosphate Buffered Saline (PBS without MgCl, without CaCl)

BioSafety I Epithelial cell culture at ≥ 80% confluence

24 well cell culture cluster plates with flat bottom and lid (Corning Inc. Costar #3524)

Sterile P-1000 pipet and tips (place in BSC during UV light sterilization)

Sterile 15 ml Corning orange capped tubes

BIOTECHNOLOGY I DNA TYPING


LAB 16

HUMAN DNA TYPING USING PCR

TIMELINE

DAY 1: Cheek cell DNA will first be isolated. The PCR reactions will be set up and

amplified in a thermal cycler. (1.5% agarose gel may be cast, if time.)

DAY 2: A 1.5% agarose gel will be cast. PCR reactions will be run, stained,

photographed and analyzed.

MATERIALS FOR PCR (DAY 1):

D1S80 primer mix (label as primer

mix)

Tris buffer (label as Tris buffer)

Chelating agent (label as resin)

10x PBS (label as 10x PBS)

200 base pair marker DNA (label as 200

bp marker)

PCR Tubes with beads that contain:

dNTP nucleotide mixture

Taq DNA polymerase buffer

Taq DNA polymerase

MgCl2

20 mL graduated cylinder

200 mL graduated cylinder (to measure

150 mL)

250 mL beaker

1.5 mL microcentrifuge tubes

1.5 mL conical tubes with screw caps

15 mL conical tubes with screw caps

racks for 15 mL tubes

sterile swabs- NOT BUCCAL SWABS!

Thermal cycler

Microcentrifuges

Vortex mixers

Centrifuge and rotor that accommodates

15 mL conical tubes

Automatic micropipetters and tips

Tips for PCR with aerosol barrier

Beaker for used tips

Heating block at 100 C (1.5 mL tubes)

dH2O

10 mL serological pipettes

electric pipet pump

250 mL flasks or beakers

ice buckets and ice

MATERIALS FOR GEL ELECTROPHORESIS (DAY 2)

Sharpie marking pen

gloves

250 ml flask

balance, spatula and weigh boats

agarose

microwave oven

heating block set at 50 C (1.5 mL tubes)

hot gloves

1x TAE electrophoresis buffer (dilute

from 50x, if needed)

100 ml graduated cylinder

dH2O

horizontal gel electrophoresis rig

automatic micropipetters and tips

used tip container

Kim wipes

1.5 ml microcentrifuge tubes

microfuge tube rack

D.C. power supply

Documents

FALL 2009/SPRING 2010 PREP GUIDE BIO:219 BIOTECHNOLOGY Iusers.stlcc.edu/departments/fvbio/Bio219_Lab_Manual/… · · 2010-01-12prep guide bio:219 biotechnology i table of contents