GeneClip™ U1 Hairpin Cloning Systems

T e c h n i c a l M a n u a l

GeneClip™ U1 HairpinCloning SystemsINSTRUCTIONS FOR USE OF PRODUCTS C8750, C8760, C8770, C8780AND C8790.

PRINTED IN USA.Revised 3/11 Part# TM256

tm256.0311:EIVD_TM.qxd 4/7/2011 1:28 PM Page 1

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM256Revised 3/11 Page 1

1. Description ..........................................................................................................2

2. Product Components and Storage Conditions ............................................5

3. Oligonucleotide Design ...................................................................................6A. siRNA Hairpin Target Sequence Selection.......................................................6B. GeneClip™ Hairpin Oligonucleotide Design ..................................................6

4. Cloning a Hairpin Insert into the pGeneClip™ Vectors...........................8A. Oligonucleotide Dilution and Annealing .........................................................8B. Ligation of a Hairpin Insert into the pGeneClip™ Vectors ..........................8C. Transformation of E. coli with

pGeneClip™ Vectors Containing Inserts ...........................................................10D. Purifying Recombinant Plasmid DNA............................................................12E. Screening for Inserts Using Pst I Digestion....................................................12

5. Transfection of pGeneClip™ Vector Constructs in an siRNA Suppression Assay...................................................................13A. Transient Transfection of the pGeneClip™ Vector Constructs ..................13B. Stable Transfection of the pGeneClip™

Puromycin, Hygromycin and Neomycin Vector Constructs ......................14C. Quantitating siRNA Target Gene Suppression .............................................15

6. Troubleshooting...............................................................................................16

7. References .........................................................................................................17

8. Appendix ...........................................................................................................19A. pGeneClip™ Vector Maps and Sequence Reference Points ........................19B. pGeneClip™ Basic Vector Restriction Enzyme Sites ....................................24C. pGeneClip™ Puromycin Vector Restriction Enzyme Sites .........................26D. pGeneClip™ Hygromycin Vector Restriction Enzyme Sites.......................28E. pGeneClip™ Neomycin Vector Restriction Enzyme Sites...........................30F. pGeneClip™ hMGFP Vector Restriction Enzyme Sites ...............................32G. Composition of Buffers and Solutions ............................................................34H. Related Products.................................................................................................34

GeneClip™ U1 Hairpin Cloning Systems

All technical literature is available on the Internet at www.promega.com/tbs Please visit the web site to verify that you are using the most current version of this

Technical Manual. Please contact Promega Technical Services if you have questions on useof this system. E-mail [email protected].


1. Description

RNA interference (RNAi), a phenomenon in which double-stranded RNAsuppresses expression of a target protein by stimulating specific degradation ofthe target mRNA, provides a powerful genetic tool for selectively silencinggene expression in many eukaryotes (1–6). In mammalian systems, shortinterfering RNAs (siRNAs) are the main effectors of the RNAi process (7,8). Thesequence-specific RNAi effect can be observed by introduction of siRNAs intocells either via transfection or by endogenous expression of 21–23 basetranscripts (8–10).

In vivo expression of siRNAs can be effectively achieved by generating DNAvectors containing a U1 RNA polymerase promoter, a template fortranscription of an siRNA and a transcription terminator sequence, thentransfecting these into eukaryotic cells. In cells, RNA polymerase II normallyrecognizes the U1 promoter to transcribe small nuclear RNAs. The U1 promoterhas been used successfully to generate siRNAs in mammalian cells (11).

GeneClip™ U1 Hairpin Cloning Systems

In the GeneClip™ U1 Hairpin Cloning Systems, siRNAs are expressed as fold-back stem-loop structures that are transcribed from the U1 promoter. TheGeneClip™ U1 Hairpin Cloning Systems include linearized plasmids(pGeneClip™ Vectors) designed for easy and fast cloning of hairpin targetsequences to allow expression of siRNA target sequences in human cells. ThepGeneClip™ Vectors contain the human U1 promoter, which allowstranscription of the hairpin target sequences and generation of hairpin siRNAin vivo. The GeneClip™ U1 Hairpin Cloning System—Basic is intended fortransient suppression of the gene of interest. The GeneClip™ U1 HairpinCloning System—hMGFP enables easy determination of transfection efficiencyand allows selection of transfected cells by fluorescence-activated cell sorting(FACS®; 12–14). The other GeneClip™ U1 Hairpin Cloning Systems offerantibiotic marker options (neomycin, hygromycin or puromycin) for selectingstably transfected eukaryotic cells so that experimental results do not dependon transfection efficiency.

Hairpin Insert

Two DNA oligonucleotides, supplied by the user, are synthesized and annealedto form a DNA insert that contains the hairpin siRNA target sequence. Uponannealing, the oligonucleotides form ends that are compatible with the ends ofthe linearized pGeneClip™ Vector and facilitate a “sticky end” ligation (Figure1). All of the pGeneClip™ Vectors contain the Ampr gene, which confersresistance to ampicillin and allows selection in E. coli.

Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM256 Printed in USA.Page 2 Revised 3/11


Screening for Successful Ligation

The GeneClip™ U1 Hairpin Cloning Systems are designed to allow easydetermination of successful ligation. Successful ligation of the annealedoligonucleotides and the vector results in creation of a second Pst I restrictionsite in addition to the Pst I site already present in the pGeneClip™ Vectors. Thepresence of an insert can be confirmed by Pst I digestion; clones containing aninsert will produce two bands on an agarose gel.

Features

More Vector Choices: These systems provide vectors containing a variety ofeukaryotic, antibiotic-selectable markers for stable transfection, or hMGFP fordetermination of transfection efficiency and selection of transfected cells byFACS® analysis.

Time Savings: Vectors are supplied predigested to eliminate time-consumingvector preparation.

Convenience: The system includes T4 DNA Ligase, 2X Rapid Ligation Buffer,Nuclease-Free Water and Oligo Annealing Buffer in addition to thepGeneClip™ Vector.

Easier Identification of Desired Clones: A Pst I digestion quickly identifiespositive recombinants.



Figure 1. Overview of the GeneClip™ U1 Hairpin Cloning System protocol.


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2. Product Components and Storage Conditions

Product Cat.#GeneClip™ U1 Hairpin Cloning System—Basic(a,b,c) C8750Each system contains sufficient reagents for 20 ligation reactions.

• 200µl 2X Rapid Ligation Buffer• 100 units T4 DNA Ligase• 1.2µg pGeneClip™ Basic Vector, 50µg/ml• 1ml Oligo Annealing Buffer• 1.25ml Nuclease-Free Water

Product Cat.#GeneClip™ U1 Hairpin Cloning System—Puromycin(a,b,c) C8760Each system contains sufficient reagents for 20 ligation reactions.

• 200µl 2X Rapid Ligation Buffer• 100 units T4 DNA Ligase• 1.2µg pGeneClip™ Puromycin Vector, 50µg/ml• 1ml Oligo Annealing Buffer• 1.25ml Nuclease-Free Water

Product Cat.#GeneClip™ U1 Hairpin Cloning System—Hygromycin(a,b,c) C8770Each system contains sufficient reagents for 20 ligation reactions.

• 200µl 2X Rapid Ligation Buffer• 100 units T4 DNA Ligase• 1.2µg pGeneClip™ Hygromycin Vector, 50µg/ml• 1ml Oligo Annealing Buffer• 1.25ml Nuclease-Free Water

Product Cat.#GeneClip™ U1 Hairpin Cloning System—Neomycin(a,b,c) C8780Each system contains sufficient reagents for 20 ligation reactions.

• 200µl 2X Rapid Ligation Buffer• 100 units T4 DNA Ligase• 1.2µg pGeneClip™ Neomycin Vector, 50µg/ml• 1ml Oligo Annealing Buffer• 1.25ml Nuclease-Free Water



Product Cat.#GeneClip™ U1 Hairpin Cloning System—hMGFP(a–g) C8790Each system contains sufficient reagents for 20 ligation reactions.

• 200µl 2X Rapid Ligation Buffer• 100 units T4 DNA Ligase• 1.2µg pGeneClip™ hMGFP Vector, 50µg/ml• 1ml Oligo Annealing Buffer• 1.25ml Nuclease-Free Water

Storage Conditions: Store at –20°C.

3. Oligonucleotide Design

3.A. siRNA Hairpin Target Sequence Selection

Choose a 19–23 nucleotide target sequence that starts with a "G" from thecoding sequence of the gene of interest. General guidelines for target sequenceselection are continuously being developed (15,16).

Although the existing rules for siRNA selection serve as a reliable guide, they donot ensure that each selected siRNA sequence will reduce gene expression, andthe optimal target sequence may have to be determined experimentally (17).

As a negative control for RNA interference, use a nonspecific target sequence ora scrambled sequence.

3.B. GeneClip™ Hairpin Oligonucleotide Design

The GeneClip™ U1 Hairpin Cloning Systems are designed to allow easy andfast detection of successful hairpin insertion. Two hairpin oligonucleotides(oligonucleotides A and B, supplied by the user) are annealed to form adouble-stranded DNA fragment for ligation into the pGeneClip™ Vectors.(Figures 1 and 4).

The hairpin oligonucleotides used in these systems are under 60bp in length.Since detection of a 60bp insert is difficult using an agarose gel, we havedevised a method to allow detection of inserts by Pst I digestion. ThepGeneClip™ Vectors contain a single Pst I site. Insertion of the hairpinoligonucleotides creates a second Pst I site, and digestion with Pst I will yieldtwo DNA fragments in the presence of an insert. Pst I digestion ofpGeneClip™ Vectors that do not contain insert will result in linearization ofthe vector (see Section 4.E).Note: Two hairpin oligonucleotides must be ordered for each sequence tested.Standard desalting of the oligonucleotides is required prior to use; gelpurification and 5´ phosphorylation are not required.


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Oligonucleotide A

Oligonucleotide A forms the template strand for cellular RNA polymerase IIand should contain the following elements:

• Overhang sequence. This four-nucleotide sequence (TCTC) iscomplementary to the overhang of the pGeneClip™ Vectors and completesthe U1 promotor sequence.

• Hairpin sequence. Includes:Target sequence. See Section 3.A. This sequence, in conjunction with itsreverse complement, forms the double-stranded portion of the cellularsiRNA hairpin.Loop sequence. This sequence provides flexibility for RNA hairpinformation within the cell. We have tested two different loop sequences(CTTCCTGTCA and AAGTTCTCT) in our system and observedcomparable suppression levels with each. Other loop sequences reported inthe literature have not been tested (18).Target sequence reverse complement. This sequence, in conjunction with thetarget sequence, forms the double-stranded portion of the cellular RNAhairpin.

• Additional CT residues. The additional CT residues are required for theformation of the second Pst I site upon ligation of the insert. These twonucleotides also form the 3´ overhang required for successful siRNAs.

Figure 2. Structure of oligonucleotide A.

Oligonucleotide B

Oligonucleotide B has regions that are complementary to oligonucleotide A,with additional sequences for ligation into the pGeneClip™ Vectors.Oligonucleotide B should contain the following elements: • Hairpin sequence complement. This sequence is complementary to, and in

the opposite orientation from, the hairpin sequence of oligonucleotide A.• Overhang sequence. This sequence (GACGTC) is complementary to the

pGeneClip™ Vector overhang and contains an additional nucleotide toform a Pst I site upon successful ligation. This newly generated Pst I siteallows easy detection of clones with inserts.

Figure 3. Structure of oligonucleotide B. Note that the sequence is representedin the 3´ to 5´ orientation.


5´ TCTC CT 3´

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Hairpin Sequence

Loop Reverse Complement

GACGTC 5´3´

4712

MAHairpin Sequence Complement


An example of hairpin oligonucleotide sequences using the 19-nucleotidetarget mRNA sequence, 5´ ggccuuucacuacuccuac 3´, is shown in Figure 4. Eacholigonucleotide in this example is 57 nucleotides in length.

4. Cloning a Hairpin Insert into the pGeneClip™ Vectors

Materials to Be Supplied by the User(Solution compositions are provided in Section 8.G)• oligonucleotide A (see Section 3)• oligonucleotide B (see Section 3)• high-efficiency competent cells (≥1 × 108cfu/µg)• 1.5ml polypropylene microcentrifuge tubes or 17 × 100mm polypropylene

tubes (Falcon Cat.# 2059)• LB plates with ampicillin• SOC medium

4.A. Oligonucleotide Dilution and Annealing

1. Dilute oligonucleotide A and oligonucleotide B in TE buffer or Nuclease-Free Water to a final concentration of 1µg/µl.

2. Assemble the annealing reaction as described below.

oligonucleotide A (1µg/µl) 2µloligonucleotide B (1µg/µl) 2µlOligo Annealing Buffer 46µlTotal volume 50µlThe final concentration of each hairpin oligonucleotide is 40ng/µl.

3. Heat the annealing reaction at 90°C for 3 minutes.

4. Transfer to a 37°C water bath and incubate for 15 minutes.

5. The annealed hairpin oligonucleotides can be used immediately or storedat –20°C for up to one month. If the annealed oligonucleotides are stored at–20°C, thaw them at room temperature prior to use. Avoid thawing theannealed oligonucleotides at temperatures above room temperature.

4.B. Ligation of a Hairpin Insert into the pGeneClip™ Vectors

The pGeneClip™ Vectors are provided as linearized vectors. No manipulationof the vectors is required prior to ligation.

1. Dilute the annealed hairpin oligonucleotides from Section 4.A, Step 5, justprior to assembling the ligation reaction as described below.Note: Do not store the diluted oligonucleotides.

annealed hairpin oligonucleotides 5µlNuclease-Free Water 45µlTotal volume 50µlThe final concentration of each oligonucleotide is 4ng/µl.




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2. Assemble the ligation reactions as described below. See Notes a and b.

Negative Control Standard(Minus Insert) Reaction

2X Rapid Ligation Buffer 5µl 5µlpGeneClip™ Vector (50ng/µl) 1µl 1µlannealed oligonucleotides A and B (4ng/µl each) – 1µlNuclease-Free Water 3µl 2µlT4 DNA Ligase (3 units/µl) 1µl 1µlTotal volume 10µl 10µl

Notes:a. The 2X Rapid Ligation Buffer contains ATP, which degrades during

temperature fluctuations. Avoid multiple freeze-thaw cycles andexposure to frequent temperature changes by making single-use aliquotsof the buffer after the buffer is thawed for the first time. Store the aliquotsat –20°C.

b. Vortex the 2X Rapid Ligation Buffer before each use.

3. Mix the reactions by pipetting. Incubate the reactions at room temperaturefor 5 minutes. Alternatively, the reactions can be incubated for 1 hour atroom temperature or overnight at 4°C.

4.C. Transformation of E. coli with pGeneClip™ Vectors Containing Inserts

The ligation of fragments with a hairpin can be inefficient, so it is essential touse competent cells with a transformation efficiency of ≥1 × 108cfu/µg DNA inorder to obtain a reasonable number of colonies. Transformation efficiency canbe confirmed by performing a control transformation reaction using a knownquantity of supercoiled plasmid DNA, typically 0.1ng, then calculating thenumber of colony forming units per microgram of DNA.

We recommend using high-efficiency JM109 Competent Cells (Cat.# L2001).Other host strains, such as DH5α™, may be used. If you are using competentcells other than JM109 Competent Cells purchased from Promega, be sure tofollow the appropriate transformation protocol. Select transformants onLB/ampicillin plates. For best results, do not use plates that are more than 1month old.Note: The use of plates containing IPTG and X-gal is not recommended.Vectors containing insert may produce blue colonies. Therefore, blue/whitescreening is not appropriate.

1. Prepare two LB/ampicillin plates for each ligation reaction and control trans-formation. Equilibrate the plates to room temperature prior to plating cells.

2. Remove the frozen, high-efficiency Competent Cells from –70°C storageand place in an ice bath until just thawed (about 5 minutes). Mix the cellsby gently flicking the tube.



3. For each ligation reaction and each transformation control, carefullytransfer 50µl of cells into a sterile 1.5ml microcentrifuge tube on ice.Note: In our experience, using larger (17 × 100mm) polypropylene tubes(e.g., Falcon Cat.# 2059) increases transformation efficiency. Tubes fromsome manufacturers bind DNA, thereby decreasing the colony number,and should be avoided.

4. Briefly centrifuge the tubes containing the ligation reactions to collectcontents at the bottom of the tube. Add 2µl of each ligation reaction to atube prepared in Step 3.To perform a transformation control, add 0.1ng of supercoiled plasmidDNA to one of the tubes prepared in Step 3. Note: The pGeneClip™ Vectors are supplied linearized and are notappropriate as a transformation control.

5. Gently flick the tubes to mix and place them on ice for 20 minutes.

6. Heat-shock the cells for 45–50 seconds in a water bath at 42°C. Do not shake.

7. Immediately return the tubes to ice for 2 minutes.

8. Add 950µl of room-temperature SOC medium to each tube. LB broth maybe substituted, but the number of colonies may be lower.

9. Incubate the tubes at 37°C with shaking (approximately 150rpm) for 1.5hours.

10. Plate 50µl of each transformation onto duplicate LB/ampicillin plates. If ahigher number of colonies is desired, pellet the cells by centrifugation at1,000 × g for 10 minutes, resuspend the cells in 200µl of SOC medium andplate 50µl of cells on each of 2 plates.

11. Incubate the plates overnight (16–24 hours) at 37°C. In our experience, atleast 100 colonies per plate are routinely seen when using competent cellsthat are 1 × 108cfu/µg DNA if 50µl is plated.

Notes: 1. The negative control ligation reaction allows determination of the number

of background colonies resulting from the GeneClip™ Vector alone. Asuccessful ligation reaction with insert typically yields at least 50 timesmore colonies than the negative control ligation reaction. The efficiency ofligation will depend upon the hairpin oligonucleotide sequence.

2. The transformation efficiency of the competent cells can be determinedusing the following equation:Equation for Transformation Efficiency (cfu/µg)

colonies on control plate 1 × 103ngng of supercoiled plasmid DNA plated × µg



4.D. Purifying Recombinant Plasmid DNA

A standard plasmid miniprep procedure can be used for screening of inserts.The miniprep process can be both laborious and time-consuming, particularlywhen large numbers of minipreps are required. A convenient and reliablemethod is the Wizard® Plus SV Minipreps DNA Purification System (Cat.#A1330).

Plasmid purification protocols that reduce the amount of endotoxin in theDNA preparation are preferred to minimize the toxic effects of endotoxins onthe cells during transfection.

4.E. Screening for Inserts Using Pst I Digestion

The pGeneClip™ Vectors contain a single Pst I site. A second Pst I site iscreated upon insertion of the hairpin oligonucleotides, and digestion with therestriction enzyme Pst I will yield two DNA fragments. Pst I digestion ofpGeneClip™ Vectors that do not contain insert will result in linearization ofthe vector (see Table 1).

Table 1. Sizes of Pst I DNA Fragments Produced by Digestion of thepGeneClip™ Vectors in the Absence and Presence of an Insert.

Pst I Fragments of Pst I Fragments of Vector Vector Without Insert Vector With Insert1

pGeneClip™ Basic Vector 3,402bp 2,461bp + 991bppGeneClip™ Puromycin Vector 4,561bp 3,209bp + 1,402bppGeneClip™ Hygromycin Vector 4,989bp 3,892bp + 1,147bppGeneClip™ Neomycin Vector 4,758bp 3,817bp + 991bppGeneClip™ hMGFP Vector 5,267bp 4,120bp + 1,197bp1The size of the larger Pst I fragment may vary with the size of the hairpin oligonucleotideinsert. The fragments sizes given here were determined using annealed hairpin oligo-nucleotides with a double-stranded region of 50bp.



5. Transfection of pGeneClip™ Vector Constructs in an siRNA Suppression Assay

Once the annealed hairpin oligonucleotides are ligated to the appropriatepGeneClip™ Vector, the resulting constructs can be used for transientexpression or for stable transfection. pGeneClip™ Vectors that contain aselectable marker (pGeneClip™ Neomycin, Hygromycin or Puromycin Vectors)can be used for stable expression of a pool of cells or individual clones.Transfection of DNA into human cells may be mediated by cationic lipids,calcium phosphate, DEAE-Dextran, polybrene-DMSO or electroporation.

5.A. Transient Transfection of the pGeneClip™ Vector Constructs

High transfection efficiency is essential for achieving substantial suppressionlevels using a transient transfection approach. Prior to testing the inhibition,optimize the transfection conditions for maximum efficiency in the system tobe tested (see below). The optimal conditions will vary with cell type,transfection method used and the amount of DNA. When using thepGeneClip™ Basic, Puromycin, Hygromycin or Neomycin Vectors,optimization can be performed using a GFP reporter such as the MonsterGreen® Fluorescent Protein phMGFP Vector (Cat.# E6421). The pGeneClip™hMGFP Vector already contains the GFP reporter. The GFP reporter can alsobe used to determine transfection efficiency for the assay. To test theeffectiveness of the pGeneClip™ Vector constructs (screening varioussequences for levels of inhibition), the use of a reporter, such as GFP, is highlyrecommended. This control can be performed as a separate transfection todetermine the percentage of the cell population transfected or as acotransfection where flow cytometry can be used to sort GFP-positive cells.The level of target RNA suppression in transfected cells can then bedetermined by taking the transfection efficiency into account.

Obtaining maximum suppression requires optimizing specific assayconditions. We have observed variations in suppression efficiency as a resultof the cell line, cell culture conditions, target sequence and transfectionconditions. Varying the amount of transfection reagent, amount of DNA andcell density can influence transfection efficiency. Obtaining the highesttransfection efficiency with low toxicity is essential for maximizing the siRNAinterference (suppression) effect in a transient assay. Additionally, maintaininghealthy cell cultures is essential for this application. The key considerations arediscussed more fully below.

Cell Density (Confluence) at Transfection

The recommended cell density for most cell types at transfection isapproximately 30–50%; this level is lower than standard transfectionexperiments where cells are plated at 50–70% confluency. The optimal celldensity should be determined for each cell type. Maintaining a dividing cellculture is essential because effective gene suppression requires proliferatingcells. Continued proliferation and the need to passage cells should beconsidered when determining the number of cells to plate.



Cell Proliferation

Successful suppression of gene expression requires actively proliferating anddividing cells, so maintaining healthy cell cultures is essential for thisapplication. It is essential to minimize the decrease in cell growth associatedwith nonspecific transfection effects and to maintain cell culture undersubconfluent conditions to assure rapid cell division. We recommend using theCellTiter-Glo® Luminescent Cell Viability Assay (Cat.# G7570) to monitor cellviability and growth.

Time

The optimal time after transfection for analyzing interference effects must bedetermined empirically by testing a range of incubation times. Typically littleinhibition is seen after 24 hours, but the maximal suppression time can varyfrom 48 to 96 hours depending on the cells used and the experimental targetstested.

5.B. Stable Transfection of the pGeneClip™ Puromycin, Hygromycin and Neomycin Vector Constructs

Note: The pGeneClip™ Basic and hMGFP Vectors are not suitable for stabletransfection.

For stable expression, antibiotic selection must be applied following transfection.Cell lines vary in the level of resistance to antibiotics, so the level of resistance ofa particular cell line must be tested before attempting stable selection of the cells.A “kill curve” will determine the minimum concentration of the antibioticneeded to kill nontransfected cells. The antibiotic concentration for selection willvary depending on the cell type and the growth rate. In addition, cells that areconfluent are more resistant to antibiotics, so it is important to keep the cellssubconfluent. The typical effective ranges and lengths of time needed forselection are given in Table 2.

Table 2. Typical Conditions for Selection of Stable Transfectants.

Effective Time NeededpGeneClip™ Vector Antibiotic Concentration For SelectionpGeneClip™ Puromycin Vector Puromycin 1–10µg/ml 2–7 dayspGeneClip™ Hygromycin Vector Hygromycin 100–1,000µg/ml 3–10 dayspGeneClip™ Neomycin Vector G-418 100–1,000µg/ml 3–14 days

For example, to generate a kill curve for G-418 selection, test G-418concentrations of 0, 100, 200, 400, 600, 800 and 1,000µg/ml in the media todetermine the concentration that is toxic to nontransfected cells. The miminumconcentration of antibiotic that kills 100% of the cells should be used insubsequent experiments.

Once the effective concentration of antibiotic has been determined, transfectedcells can be selected for resistance.



1. Following transfection, seed the cells at a low cell density.

2. Apply antibiotic to the medium at the effective concentration determinedfrom the kill curve.

3. Prepare a control plate for all selection experiments by treatingnontransfected cells with antibiotic in medium under the experimentalconditions. This control plate will confirm whether the conditions ofantibiotic selection were sufficiently stringent to eliminate cells notexpressing the resistance gene.

4. Change the medium every 2–3 days until drug-resistant clones appear.

5. Once clones (or pools of cells) are selected, grow the cells in mediacontaining the antibiotic at a reduced antibiotic concentration, typically25–50% of the level used during selection.

5.C. Quantitating siRNA Target Gene Suppression

Reduction of the targeted gene expression can be measured by 1) monitoringphenotypic changes of the cell, 2) measuring changes in mRNA levels (e.g.,using RT-PCR), or 3) detecting changes in protein level by immuno-cytochemistry or Western blot analysis (19–22). The suppression effect will varydepending on the target, cell line and experimental conditions.

Controlling for nonspecific effects on other targets is very important. As anegative control, cells can be transfected with either a nonspecific or scrambledtarget sequence. This will show that suppression of gene expression is specific tothe expression of the hairpin siRNA target sequences. When suppression isdetermined by Western analysis, positive controls for other genes (e.g., tubulinor actin) should be included. Additional details can be found in reference 23.

Using the GeneClip™ hMGFP Vector, expression of hMGFP allowsnormalization of target gene suppression to transfection efficiency.Alternatively, the suppressive effects can be analyzed only in transfected cells byseparating those cells by FACS® analysis or by analyzing target genesuppression at the individual cell level. Peak excitation of hMGFP occurs at505nm, with a shoulder at 480nm, and peak emission occurs at 515nm. hMGFPexpression can be monitored by fluorescence microscopy using an excitationfilter of 470±20nm (470/40nm) and an emission filter of 515nm (long pass).

The psiCHECK™-1 and -2 Vectors (Cat.# C8011, C8021) can also be used tomeasure target gene suppression. These Vectors enable the monitoring ofchanges in expression of a target gene fused to a reporter, Renilla luciferase,gene. In these vectors, the gene of interest is cloned into the multiple cloningregion located downstream of the Renilla translational stop codon. Initiation ofthe RNAi process towards a gene of interest results in cleavage andsubsequent degradation of the fusion mRNA. Measurement of decreasedRenilla luciferase activity is a convenient indicator of RNAi effect (24).



6. Troubleshooting

Symptoms Causes and Comments

Low number or Ligation reaction failed. Ligation reactions withno colonies insert typically yield at least 50 times more

colonies than negative control reactions. If the number of colonies is the same as the negative control, this indicates a problem with the insertor the ligation reaction.

The 2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-thaw cycles by making single-use aliquots of the buffer. Usea fresh vial of buffer.

Incorrect oligonucleotide sequences. Oligonucleotide A must have an overhang of TCTC, and oligonucleotide B must have an overhang of CGTC. See Figure 4.

Improper dilution of the 2X Rapid Ligation Buffer. The Rapid Ligation Buffer is provided ata 2X concentration. Use 5µl in a 10µl reaction.

Ineffective transformation or poor-quality competent cells. Perform a control trans-formation with supercoiled plasmid DNA to ensure that the efficiency of the competent cells is ≥1 × 108cfu/µg DNA (see Section 4.C). The pGeneClip™ Vectors are supplied linearized and are not appropriate as transformation controls.

Oligonucleotides failed to anneal. Confirm oligonucleotide sequences. Repeat annealing step and use annealed oligonucleotides immediately.

Insert not present in Poor-quality oligonucleotides. Try HPLC-pGeneClip™ Vector purified or gel-purified oligonucleotides.

Ligation reaction failed. Ligation reactions with insert typically yield at least 50 times more colonies than negative control reactions. If the number of colonies is the same as the negative control, this indicates a problem with the insert or the ligation reaction.

The 2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-thaw cycles by making single-use aliquots of the buffer. Usea fresh vial of buffer.



Symptoms Causes and Comments

Insert not present in Incorrect oligonucleotide sequences.pGeneClip™ Vector Oligonucleotide A must have an overhang(continued) of TCTC, and oligonucleotide B must have an

overhang of CGTC. See Figure 4.

No suppression or Ineffective siRNA target sequence. Test at leastlow-level suppression of 3–6 target sequences for each mRNA to identify target gene the sequences that result in the highest level of

suppression.

Time point not optimal. Assay cells at several time points within 2–6 days after transfection todetermine the peak effect.

Low transfection efficiency. Use a GFP vector, such as the Monster Green® Fluorescent Protein phMGFP Vector (Cat.# E6421), to determine transfection efficiency. If the efficiency is low, optimize transfection conditions, as described inSection 5.

Inadequate detection. If reduction of the targeted gene is analyzed by immunocyto-chemistry or Western blot, check for antibody specificity. Include controls (e.g., actin or tubulin) in your analysis (20).

Cell growth or viability affected by specific target sequence. Transfection of the vector may affect cell proliferation when compared to nontransfected cells. We recommend monitoring cell viability and growth to account for any changes in cell number during the suppression assay.

7. References

1. Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. Cell 101, 235–8.

2. Zamore, P.D. (2001) RNA interference: Listening to the sound of silence. Nat. Struct.Biol. 8, 746–50.

3. Sharp, P.A. (2001) RNA interference–2001. Genes Dev. 15, 485–90.

4. Hutvagger, G. and Zamore, P.D. (2002) RNAi: Nature abhors a double strand. Curr.Opin. Genet. Dev. 12, 225–32.

5. Dykxhoorn, D.M., Novina, C.D. and Sharp, P.A. (2003) Killing the messenger: ShortRNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457–67.

6. Denli, A.M. and Hannon, G.J. (2003) RNAi: An ever-growing puzzle. Trends Biochem.Sci. 28, 196–201.



7. References (continued)

7. Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) RNA interference is mediated by21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200.

8. Yu, J-Y. et al. (2002) RNA interference by expression of short-interfering RNA andhairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047–52.

9. Sui, G. et al. (2002) A DNA vector-based RNAi technology to suppress geneexpression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–20.

10. Brummelkamp, T.R., Bernards, R. and Agami, R. (2002) A system for stableexpression of short interfering RNAs in mammalian cells. Science 296, 550–3.

11. Novarino, G. et al. (2004) Involvement of the intracellular ion channel CLIC1 inmicroglia-mediated β-amyloid-induced neurotoxicity. J. Neurosci. 24, 5322–30.

12. Cormack, B.P., Valdivia, R.H. and Falkow, S. (1996) FACS-optimized mutants of thegreen fluorescent protein (GFP). Gene 173, 33–8.

13. Sorensen, T.U. et al. (1999) Safe sorting of GFP-transduced live cells for subsequentculture using a modified FACS vantage. Cytometry 37, 284–90.

14. Galbraith, D.W. et al. (1999) Flow cytometric analysis and FACS sorting of cells basedon GFP accumulation. Methods Cell. Biol. 58, 315–41.

15. Khvorova, A. et al. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell115, 209–16.

16. Ui-Tei, K. et al. (2004) Guidelines for the selection of highly effective siRNA sequencesfor mammalian and chick RNA interference. Nucl. Acid Res. 32, 936–48.

17. Vidugiriene, J. et al. (2004) The use of bioluminescent reporter genes for RNAioptimization. Promega Notes 87, 2–6.

18. Elbashir, S.M. et al. (2002) Analysis of gene function in somatic mammalian cellsusing small interfering RNAs. Methods 26, 199–213.

19. Xia, H. et al. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotech.20, 1006–10.

20. Huang, Y. et al. (2003) Erbin suppresses the MAP kinase pathway. J. Biol. Chem. 278,1108–14.

21. Kullmann, M. et al. (2002) ELAV/Hu proteins inhibit p27 translation via an IRESelement in the p27 5´UTR. Genes Dev. 16, 3087–99.

22. Lang, V. et al. (2003) βTrCP-mediated proteolysis of NF-κB p105 requiresphosphorylation of p105 series 927 and 932. Mol. Cell. Biol. 23, 402–13.

23. Editorial (2003) Whither RNAi? Nature Cell Biol. 5, 489–90.

24. Kumar, R. et al. (2003) High-throughput selection of effective RNAi probes for genesilencing. Genome Res. 13, 2333–40.



8. Appendix

8.A. pGeneClip™ Vector Maps and Sequence Reference Points

The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listedlocations of the vector features and restriction enzyme sites are in relation tobase 1 (the T7 RNA polymerase transcription initiation site) and are numberedas though the overhangs had been filled in and then ligated.

Figure 5. pGeneClip™ Basic Vector circle map and sequence reference points.

pGeneClip™ Basic Vector Sequence Reference PointsT7 RNA polymerase transcription initiation site 1U1 promoter (human –392 to +1) 46–43810bp spacer 439–448U1 termination sequence 449–465SP6 RNA polymerase promoter (–17 to +3) 527–546SP6 RNA polymerase promoter binding site 529–547Binding site of pUC/M13 Reverse Sequencing Primer 564–585β-lactamase (Ampr) coding region 1724–2584Binding site of pUC/M13 Forward Sequencing Primer 3336–3359T7 RNA polymerase promoter (–17 to +3) 3386–3


4790

MA

Ampr

U1 p

romoter

linearized pGeneClip™Basic Vector

(3,402bp)

T7

➞

SP6➞

AGAG

1 start

529Pst I 1432

GCAG


Figure 6. pGeneClip™ Puromycin Vector circle map and sequence referencepoints.

pGeneClip™ Puromycin Vector Sequence Reference PointsT7 RNA polymerase transcription initiation site 1U1 promoter (human –392 to +1) 46–43810bp spacer 439–448U1 termination sequence 449–465SP6 RNA polymerase promoter (–17 to +3) 527–546SP6 RNA polymerase promoter binding site 529–547Binding site of pUC/M13 Reverse Sequencing Primer 564–585SV40 enhancer and early promoter 798–1216SV40 minimum origin of replication 1114–1179Puromycin-N-acetyltransferase coding region 1239–1838Synthetic polyadenylation signal 1883–1931β-lactamase (Ampr) coding region 2883–3743Binding site of pUC/M13 Forward Sequencing Primer 4495–4518T7 RNA polymerase promoter (–17 to +3) 4545–3


4791

MA

Ampr

U1 p

romoter

linearized pGeneClip™Puromycin Vector

(4,561bp)

T7

➞

SP6➞

AGAG

GCAG

1 start

529Pst I1843

SV40 Enhancer/Early Promoter

SyntheticPolyadenylation

Signal

Puromycin


Figure 7. pGeneClip™ Hygromycin Vector circle map and sequence referencepoints.

pGeneClip™ Hygromycin Vector Sequence Reference PointsT7 RNA polymerase transcription initiation site 1U1 promoter (human –392 to +1) 46–43810bp spacer 439–448U1 termination sequence 449–465SP6 RNA polymerase promoter (–17 to +3) 527–546SP6 RNA polymerase promoter binding site 529–547Binding site of pUC/M13 Reverse Sequencing Primer 564–585SV40 enhancer and early promoter 798–1216SV40 minimum origin of replication 1114–1179Hygromycin phosphotransferase coding region 1251–2276Synthetic polyadenylation signal 2311–2359β-lactamase (Ampr) coding region 3311–4171Binding site of pUC/M13 Forward Sequencing Primer 4923–4946T7 RNA polymerase promoter (–17 to +3) 4973–3


4789

MA

Ampr

U1 p

romoter

linearized pGeneClip™Hygromycin Vector

(4,989bp)

T7

SP6

AGAG

GCAG

1 start

529Pst I1588



Signal

Hygromycin

➞


Figure 8. pGeneClip™ Neomycin Vector circle map and sequence referencepoints.

pGeneClip™ Neomycin Vector Sequence Reference PointsT7 RNA polymerase transcription initiation site 1U1 promoter (human –392 to +1) 46–43810bp spacer 439–448U1 termination sequence 449–465SP6 RNA polymerase promoter (–17 to +3) 527–546SP6 RNA polymerase promoter binding site 529–547Binding site of pUC/M13 Reverse Sequencing Primer 564–585SV40 enhancer and early promoter 798–1216SV40 minimum origin of replication 1114–1179Neomycin phosphotransferase coding region 1251–2045Synthetic polyadenylation signal 2080–2128β-lactamase (Ampr) coding region 3080–3940Binding site of pUC/M13 Forward Sequencing Primer 4692–4715T7 RNA polymerase promoter (–17 to +3) 4742–3


4788

MA

Ampr

U1 p

romoter

linearized pGeneClip™Neomycin Vector

(4,758bp)

T7

➞

SP6➞

AGAG

GCAG

1 start

529Pst I1432



Signal

Neomycin


Figure 9. pGeneClip™ hMGFP Vector circle map and sequence reference points.

pGeneClip™ hMGFP Vector Sequence Reference PointsT7 RNA polymerase transcription initiation site 1U1 promoter (human –392 to +1) 46–43810bp spacer 439–448U1 termination sequence 449–465SP6 RNA polymerase promoter (–17 to +3) 527–546SP6 RNA polymerase promoter binding site 529–547Binding site of pUC/M13 Reverse Sequencing Primer 564–585CMV enhancer/promoter 801–1550Chimeric intron 1690–1822hMGFP open reading frame 1880–2563Synthetic polyadenylation signal 2589–2637β-lactamase (Ampr) coding region 3589–4449Binding site of pUC/M13 Forward Sequencing Primer 5201–5224T7 RNA polymerase promoter (–17 to +3) 5250–3


4792

MA

Ampr

U1 p

romoter

linearized pGeneClip™hMGFP Vector

(5,267bp)

hMGFPCMVPromoter

Pst I1638

Intron

T7

➞

SP6➞

AGAG

GCAG

1 start

529


8.B. pGeneClip™ Basic Vector Restriction Enzyme Sites

The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed locationsof the restriction enzyme sites are in relation to base 1 (the T7 RNA polymerasetranscription initiation site). The following restriction enzyme tables were constructedusing DNASTAR® sequence analysis software. Please note that we have not verifiedthis information by restriction digestion with each enzyme listed. The location givenspecifies the 3´ end of the cut DNA (the base to the left of the cut site). For moreinformation on the cut sites of these enzymes, or if you identify a discrepancy, pleasecontact your local Promega Branch or Distributor. In the U.S., contact PromegaTechnical Services at 800-356-9526. Vector sequences are also available in theGenBank® database (GenBank®/EMBL Accession Number AY744385) and on theInternet at: www.promega.com/vectors/

Table 3. Restriction Enzymes That Cut the pGeneClip™ Basic Vector 1–5 Times.


Enzyme # of Sites LocationAat II 1 112 Acc I 2 29, 459 Acy I 2 109, 2334 Afl III 2 502, 905 Alw26 I 2 1858, 2634 Alw44 I 2 1219, 2464 AlwN I 2 242, 1321Apa I 1 14 AspH I 4 497, 1223, 2383, 2468Ava I 2 357, 487 Ava II 4 50, 305, 1935, 2157 BamH I 1 40 Ban I 3 649, 1745, 3028 Ban II 4 14, 358, 497, 3066 Bgl I 2 1917, 3235 Bgl II 1 432Bsa I 1 1858 BsaA I 1 2991 BsaH I 2 109, 2334 Bsp120 I 1 10 BspH I 2 1624, 2632 BssS I 2 1078, 2461 BstX I 1 506 BstZ I 1 468 Bsu36 I 1 46 Cfr10 I 2 1877, 3092 Cla I 1 476 Dde I 5 46, 1180, 1588, 1754,

2294 Dra I 3 1663, 1682, 2374 Dra III 1 2991 Drd I 2 1013, 2946

Enzyme # of Sites LocationEae I 4 468, 744, 2185, 3372Eag I 1 468 Ear I 3 789, 2592, 3280 EclHK I 1 1797 Eco52 I 1 468 Eco81 I 1 46 EcoICR I 1 495 EcoR I 1 34 EcoR V 1 18 Fok I 5 522, 1763, 1944,

2231, 3318 Fsp I 2 2019, 3242 Hae II 4 783, 1153, 3142,

3150 Hga I 5 84, 1016, 1593,

2323, 3208 Hinc II 2 30, 483 Hind II 2 30, 483 Hpa I 1 483 Hsp92 I 2 109, 2334 Mae I 5 23, 1400, 1652,

1987, 3142 Mlu I 1 502Nae I 1 3094 Nci I 3 1285, 1980, 2331 NgoM IV 1 3092 Not I 1 468 Nsi I 1 515 Nsp I 1 909 PaeR7 I 1 487 Ppu10 I 1 511 Pst I* 1 1432 .*A second Pst I site is created upon ligation of an insert.


Table 3. Restriction Enzymes That Cut the pGeneClip™ Basic Vector 1–5 Times(continued).

Table 4. Restriction Enzymes That Do Not Cut the pGeneClip™ Basic Vector.

Table 5. Restriction Enzymes That Cut the pGeneClip™ Basic Vector 6 or MoreTimes.

Note: The enzymes listed in boldface type are available from Promega.


Enzyme # of Sites LocationPvu I 2 2167, 3263 Pvu II 3 333, 729, 3292 Rsa I 1 2277 Sac I 1 497 Sal I 1 28 Sca I 1 2277 Sin I 4 50, 305, 1935, 2157

Enzyme # of Sites LocationSsp I 2 2601, 2783 Sty I 1 120 Tfi I 3 325, 740, 880 Vsp I 3 676, 735, 1969 Xba I 1 22 Xho I 1 487 Xmn I 2 277, 2396

Acc B7 IAcc IIIAcc65 IAfl IIAge IAsc IAvr IIBal IBbe IBbrP IBbs IBbu IBcl I

Blp IBpu1102 IBsaB IBsaM IBsm IBspM IBsrG IBssH IIBst1107 IBst98 IBstE IICsp ICsp45 I

Dra IIDsa IEco47 IIIEco72 IEcoN IEhe IFse IHind IIII-Ppo IKas IKpn INar INco I

Nde INhe INru IPac IPflM IPinA IPme IPml IPpuM IPshA IPsp5 IIPspA IRsr II

Sac IISfi ISgf ISgrA ISma ISnaB ISpe ISph ISpl ISrf ISse8387 IStu ISwa I

Tth111 IXcm IXma I

Aci IAlu IBbv IBsaO I BsaJ I Bsp1286 IBsr I

BsrS IBst71 IBstO IBstU ICfo IDpn IDpn II

Fnu4H IHae III Hha IHinf IHpa IIHph IHsp92 II

Mae IIMae IIIMbo IMbo IIMnl IMse IMsp I

MspA1 INde IINla III Nla IVPle ISau3A ISau96 I

ScrF ISfaN ITaq I Tru9 I Xho II


8.C. pGeneClip™ Puromycin Vector Restriction Enzyme Sites

The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed locationsof the restriction enzyme sites are in relation to base 1 (the T7 RNA polymerasetranscription initiation site). The following restriction enzyme tables were constructedusing DNASTAR® sequence analysis software. Please note that we have not verifiedthis information by restriction digestion with each enzyme listed. The location givenspecifies the 3´ end of the cut DNA (the base to the left of the cut site). For moreinformation on the cut sites of these enzymes, or if you identify a discrepancy, pleasecontact your local Promega Branch or Distributor. In the U.S., contact PromegaTechnical Services at 800-356-9526. Vector sequences are also available in theGenBank® database (GenBank®/EMBL Accession Number AY745747) and on theInternet at: www.promega.com/vectors/

Table 6. Restriction Enzymes That Cut the pGeneClip™ Puromycin Vector 1–5Times.


Enzyme # of Sites LocationAat II 3 112, 1285 1783 Acc I 3 29, 459, 1348Acc65 I 1 845 Afl III 2 502, 2063 Age I 1 1868 Alw26 I 3 1713, 3017, 3793 Alw44 I 2 2377, 3623 AlwN I 2 242, 2479 Apa I 1 14 AspH I 5 497, 1672, 2381,

3542, 3627 Ava I 4 357, 487, 1288, 1849 Avr II 1 1195 Bal I 1 1614 BamH I 2 40, 1844 Bbe I 2 1450, 1581 Bbu I 2 943, 1015 Bgl I 5 1148, 1455, 1586,

3076, 4394 Bgl II 1 432 Bsa I 2 1713, 3017 BsaA I 1 4150 Bsp120 I 1 10 BspH I 2 2783, 3791 BssH II 1 1694 BssS I 2 2236, 3620 BstE II 1 1373 BstX I 1 506 BstZ I 2 468, 1687 Bsu36 I 1 46 Cfr10 I 4 1585, 1868, 3036,

4251

Enzyme # of Sites LocationCla I 1 476 Csp I 1 1355 Csp45 I 1 1933 Dra I 4 1857, 2822, 2841,

3533 Dra III 2 1810, 4150 Drd I 2 2171, 4105 Dsa I 4 806, 1102, 1255, 1450Eag I 2 468, 1687 Ear I 4 789, 1398, 3751, 4439 EclHK I 1 2956 Eco52 I 2 468, 1687 Eco81 I 1 46 EcoICR I 1 495 EcoR I 1 34 EcoR V 1 18 Ehe I 2 1448, 1579 Fsp I 3 799, 3178, 4401 Hinc II 3 30, 483, 1349 Hind II 3 30, 483, 1349 Hind III 2 1211, 1942 Hpa I 1 483 Kas I 2 1446, 1577 Kpn I 1 849 Mlu I 1 502 Nae I 1 4253 Nar I 2 1447, 1578 Nco I 2 806, 1102 NgoM IV 1 4251 Nhe I 1 1229 Not I 1 468 Nsi I 3 515, 945, 1017


Table 6. Restriction Enzymes That Cut the pGeneClip™ Puromycin Vector 1–5Times (continued).

Table 7. Restriction Enzymes that Do Not Cut the pGeneClip™ Puromycin Vector.

Table 8. Restriction Enzymes that Cut the pGeneClip™ Puromycin Vector 6 orMore Times.



Enzyme # of Sites LocationNsp I 3 943, 1015, 2067 PaeR7 I 2 487, 1849 PinA I 1 1868 Pme I 1 1857 Ppu10 I 3 511, 941, 1013 PspA I 1 1288 Pst I* 1 1843 Pvu I 2 3326, 4422 Pvu II 4 333, 729, 871, 4451 Rsa I 4 847, 1248, 1297, 3436 Rsr II 1 1355 Sac I 1 497 Sac II 1 1453 Sal I 2 28, 1347 Sca I 1 3436

Enzyme # of Sites LocationSfi I 1 1148 Sma I 1 1290 Sph I 2 943, 1015 Spl I 1 1295 Ssp I 2 3760, 3942 Stu I 2 1194, 1570 Sty I 5 120, 806, 1102, 1195,

1591 Tfi I 4 325, 740, 1217, 2038 Tth111 I 1 1281 Vsp I 3 676, 735, 3128 Xba I 2 22, 1862 Xho I 2 487, 1849 Xma I 1 1288 Xmn I 2 277, 3555.*A second Pst I site is created upon ligation of an insert.

AccB7 IAcc IIIAfl IIAsc IBbrP IBbs IBcl I

Blp IBpu1102 IBsaB IBsaM IBsm IBspM IBsrG I

Bst1107 IBst98 IDra IIEco47 IIIEco72 IEcoN IFse I

I-Ppo INde INru IPac IPflM IPml IPpuM I

PshA IPsp5 IISgf ISgrA ISnaB ISpe ISrf I

Sse8387 ISwa IXcm I

Aci IAcy IAlu IAva IIBan IBan IIBbv IBsaO IBsaH IBsaJ I

Bsp1286 IBsr IBsrS IBst71 I BstO IBstU ICfo IDde I Dpn IDpn II

Eae IFnu4H IFok IHae IIHae IIIHga I Hha IHinf IHpa IIHph I

Hsp92 IHsp92 IIMae I Mae IIMae III Mbo IMbo IIMnl I Mse IMsp I

MspA1 INci INde IINla IIINla IVPle I Sau3A ISau96 IScrF ISfaN I

Sin ITaq ITru9 IXho II



8.D. pGeneClip™ Hygromycin Vector Restriction Enzyme Sites

The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed locationsof the restriction enzyme sites are in relation to base 1 (the T7 RNA polymerasetranscription initiation site) and are numbered as though the overhangs had beenfilled in and then ligated. The following restriction enzyme tables were constructedusing DNASTAR® sequence analysis software. Please note that we have not verifiedthis information by restriction digestion with each enzyme listed. The location givenspecifies the 3´ end of the cut DNA (the base to the left of the cut site). For moreinformation on the cut sites of these enzymes, or if you identify a discrepancy, pleasecontact your local Promega Branch or Distributor. In the U.S., contact PromegaTechnical Services at 800-356-9526. Vector sequences are also available in theGenBank® database (GenBank®/EMBL Accession Number AY745745) and on theInternet at: www.promega.com/vectors/

Table 9. Restriction Enzymes That Cut the pGeneClip™ Hygromycin Vector 1–5Times.Enzyme # of Sites LocationAat II 2 112, 1278 Acc I 2 29, 459 Acc III 3 1472, 2009, 2145 Acc65 I 1 845 Acy I 4 109, 1275, 2243, 3921Afl III 2 502, 2491 Age I 1 2296Alw26 I 2 3445, 4221 Alw44 I 4 1531, 1833, 2805, 4051 AlwN I 2 242, 2907 Apa I 1 14 Ava I 4 357, 487, 1240, 2277 Avr II 1 1195 BamH I 1 40 Ban I 5 649, 845, 1825, 3332,

4615 Ban II 4 14, 358, 497, 4653 Bbu I 2 943, 1015 Bgl I 3 1148, 3504, 4822 Bgl II 1 432 Bsa I 1 3445 BsaA I 1 4578 BsaH I 4 109, 1275, 2243, 3921 Bsp120 I 1 10 BspH I 2 3211, 4219 BspM I 1 1564 BssS I 4 1351, 1830, 2664, 4048BstX I 1 506 BstZ I 4 468, 1456, 1621, 2191 Bsu36 I 1 46 Cfr10 I 4 1589, 2296, 3464, 4679 Cla I 1 476

Enzyme # of Sites LocationCsp I 1 1659 Csp45 I 1 2361 Dra I 4 2285, 3250, 3269, 3961 Dra III 3 1539, 1832, 4578 Drd I 4 1755, 2136, 2599, 4533 Dsa I 5 806, 1102, 1603, 1959,

2028 Eag I 4 468, 1456, 1621, 2191Ear I 3 789, 4179, 4867 EclHK I 2 1313, 3384 Eco52 I 4 468, 1456, 1621, 2191 Eco81 I 1 46 EcoICR I 1 495 EcoR I 2 34, 1494 EcoR V 1 18 Fsp I 3 799, 3606, 4829 Hae II 4 783, 2739, 4729, 4737 Hinc II 3 30, 483, 2087 Hind II 3 30, 483, 2087 Hind III 2 1211, 2370 Hpa I 1 483 Hsp92 I 4 109, 1275, 2243, 3921 Kpn I 1 849 Mlu I 1 502 Nae I 1 4681 Nco I 3 806, 1102, 1603 Nde I 1 1701 NgoM IV 1 4679 Nhe I 1 1229 Not I 1 468 Nsi I 3 515, 945, 1017 Nsp I 3 943, 1015, 2495



Table 9. Restriction Enzymes That Cut the pGeneClip™ Hygromycin Vector 1–5Times (continued).

Table 10. Restriction Enzymes that Do Not Cut the pGeneClip™ HygromycinVector.

Table 11. Restriction Enzymes that Cut the pGeneClip™ Hygromycin Vector 6 orMore Times.


Enzyme # of Sites LocationPaeR7 I 2 487, 2277 PinA I 1 2296 Pme I 1 2285 Ppu10 I 3 511, 941, 1013 PshA I 1 1278 PspA I 1 1240 Pst I* 1 1588 Pvu I 4 1237, 1615, 3754,

4850 Pvu II 4 333, 729, 871, 4879 Rsa I 4 847, 2168, 2221, 3864 Rsr II 1 1659 Sac I 1 497Sac II 1 2031 Sal I 1 28 Sca I 2 2221, 3864

Enzyme # of Sites LocationSfi I 1 1148 Sgf I 2 1237, 1615 Sma I 1 1242 Sph I 2 943, 1015 Srf I 1 1242 Ssp I 2 4188, 4370 Stu I 1 1194 Sty I 5 120, 806, 1102,

1195,1603 Tth111 I 1 1755 Vsp I 3 676, 735, 3556 Xba I 2 22, 2290 Xho I 2 487, 2277 Xma I 1 1240 Xmn I 2 277, 3983

.*A second Pst I site is created upon ligation of an insert.

AccB7 IAfl IIAsc IBal IBbe IBbrP IBbs I

Bcl IBlp IBpu1102 IBsaB IBsaM IBsm IBsrG I

BssH IIBst1107 IBst98 IBstE IIDra IIEco47 IIIEco72 I

EcoN IEhe IFse II-Ppo IKas INar INru I

Pac IPflM IPml IPpuM IPsp5 IISgrA ISnaB I

Spe ISpl ISse8387 ISwa IXcm I

Aci I Alu I Ava II Bbv I BsaO I BsaJ I Bsp1286 IBsr I BsrS I

Bst71 I BstO IBstU I Cfo IDde I Dpn IDpn II Eae I Fnu4H I

Fok I Hae IIIHga I Hha I Hinf IHpa IIHph I Hsp92 IIMae I

Mae II Mae III Mbo I Mbo IIMnl I Mse I Msp I MspA1 I Nci I

Nde IINla IIINla IV Ple I Sau3A I Sau96 I ScrF I SfaN I Sin I

Taq ITfi I Tru9 I Xho II



8.E. pGeneClip™ Neomycin Vector Restriction Enzyme Sites

The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed locationsof the restriction enzyme sites are in relation to base 1 (the T7 RNA polymerasetranscription initiation site). The following restriction enzyme tables wereconstructed using DNASTAR® sequence analysis software. Please note that we havenot verified this information by restriction digestion with each enzyme listed. Thelocation given specifies the 3´ end of the cut DNA (the base to the left of the cutsite). For more information on the cut sites of these enzymes, or if you identify adiscrepancy, please contact your local Promega Branch or Distributor. In the U.S.,contact Promega Technical Services at 800-356-9526. Vector sequences are alsoavailable in the GenBank® database (GenBank®/EMBL Accession NumberAY745746) and on the Internet at: www.promega.com/vectors/

Table 12. Restriction Enzymes That Cut the pGeneClip™ Neomycin Vector 1–5Times.Enzyme # of Sites LocationAat II 1 112 Acc I 2 29, 459 Acc65 I 1 845 Acy I 3 109, 1379, 3690 Afl III 2 502, 2260 Age I 1 2065 Alw26 I 2 3214, 3990 Alw44 I 2 2574, 3820 AlwN I 2 242, 2676 Apa I 1 14 Ava I 4 357, 487, 1240, 2046 Ava II 5 50, 305, 1895, 3291,

3513 Avr II 1 1195 Bal I 1 1461 BamH I 1 40 Ban II 5 14, 358, 497, 1744,

4422Bbe I 1 1382 Bbu I 3 943, 1015, 1784 Bgl I 3 1148, 3273, 4591 Bgl II 1 432 Bsa I 1 3214 BsaA I 2 1683, 4347 BsaH I 3 109, 1379, 3690 Bsp120 I 1 10 BspH I 2 2980, 3988 BspM I 2 1266, 1647 BssH II 1 1776 BssS I 3 1971, 2433, 3817 BstX I 1 506 BstZ I 2 468, 1285

Enzyme # of Sites LocationBsu36 I 1 46 Cfr10 I 5 1698, 1879, 2065, 3233,

4448 Cla I 1 476 Csp I 1 1895 Csp45 I 1 2130 Dra I 4 2054, 3019, 3038, 3730 Dra III 1 4347 Drd I 3 1406, 2368, 4302 Dsa I 3 806, 1102, 1811 Eag I 2 468, 1285 Ear I 5 789, 1723, 1933, 3948,

4636 EclHK I 1 3153 Eco52 I 2 468, 1285 Eco81 I 1 46 EcoICR I 1 495 EcoR I 1 34EcoR V 1 18 Ehe I 1 1380 Fsp I 4 799, 1481, 3375, 4598 Hae II 5 783, 1382, 2508, 4498,

4506 Hinc II 2 30, 483 Hind II 2 30, 483 Hind III 2 1211, 2139 Hpa I 1 483 Hsp92 I 3 109, 1379, 3690 Kas I 1 1378 Kpn I 1 849 Mlu I 1 502 Nae I 2 1881, 4450



Table 12. Restriction Enzymes That Cut the pGeneClip™ Neomycin Vector 1–5Times (continued).

Table 13. Restriction Enzymes that Do Not Cut the pGeneClip™ Neomycin Vector.

Table 14. Restriction Enzymes that Cut the pGeneClip™ Neomycin Vector 6 orMore Times.


Enzyme # of Sites LocationNar I 1 1379 Nco I 3 806, 1102, 1811 NgoM IV 2 1879, 4448 Nhe I 1 1229 Not I 1 468 Nsi I 3 515, 945, 1017 Nsp I 4 943, 1015, 1784, 2264 PaeR7 I 2 487, 2046 PinA I 1 2065 Pme I 1 2054 Ppu10 I 3 511, 941, 1013 PspA I 1 1240 Pst I* 1 1432 Pvu I 3 1237, 3523, 4619 Pvu II 5 333, 729, 871, 1485,

4648 Rsa I 3 847, 1685, 3633 Rsr II 1 1895 Sac I 1 497

Enzyme # of Sites LocationSal I 1 28 Sca I 1 3633 Sfi I 1 1148 Sgf I 1 1237 Sin I 5 50, 305, 1895, 3291,

3513 Sma I 1 1242 Sph I 3 943, 1015, 1784 Srf I 1 1242 Ssp I 2 3957, 4139 Stu I 1 1194 Sty I 5 120, 806, 1102, 1195,

1811 Tth111 I 1 1497 Vsp I 3 676, 735, 3325 Xba I 2 22, 2059 Xho I 2 487, 2046 Xma I 1 1240 Xmn I 2 277, 3752 .*A second Pst I site is created upon ligation of an insert.

AccB7 IAcc IIIAfl IIAsc IBbrP IBbs IBcl I

Blp IBpu1102 IBsaB IBsaM IBsm IBsrG IBst1107 I

Bst98 IBstE IIDra IIEco47 IIIEco72 IEcoN IFse I

I-Ppo INde INru IPac IPflM IPml IPpuM I

PshA IPsp5 IISac IISgrA ISnaB ISpe ISpl I

Sse8387 ISwa IXcm I

Aci I Alu I AspH I Ban IBbv IBsaO IBsaJ IBsp1286 IBsr I

BsrS IBst71 I BstO IBstU I Cfo IDde IDpn I Dpn II Eae I

Fnu4H IFok IHae IIIHga IHha IHinf IHpa IIHph I Hsp92 II

Mae IMae II Mae IIIMbo IMbo IIMnl IMse IMsp IMspA1 I

Nci INde IINla III Nla IV Ple I Sau3A ISau96 I ScrF I SfaN I

Taq ITfi I Tru9 IXho II



8.F. pGeneClip™ hMGFP Vector Restriction Enzyme Sites

The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listedlocations of the restriction enzyme sites are in relation to base 1 (the T7 RNApolymerase transcription initiation site). The following restriction enzymetables were constructed using DNASTAR® sequence analysis software. Pleasenote that we have not verified this information by restriction digestion witheach enzyme listed. The location given specifies the 3´ end of the cut DNA (thebase to the left of the cut site). For more information on the cut sites of theseenzymes, or if you identify a discrepancy, please contact your local PromegaBranch or Distributor. In the U.S., contact Promega Technical Services at 800-356-9526. Vector sequences are also available in the GenBank® database(GenBank®/EMBL Accession Number AY744386) and on the Internet at:www.promega.com/vectors/

Table 15. Restriction Enzymes That Cut the pGeneClip™ hMGFP Vector 1–5Times.Enzyme # of Sites LocationAat II 5 112, 1078, 1131 1214,

1400AccB7 I 1 2458 Acc I 2 29, 459Afl II 2 1628, 1647 Afl III 2 502, 2769 Age I 1 2574 Alw44 I 2 3083, 4329 AlwN I 2 242, 3185Apa I 1 14Ava I 4 357, 487, 1862, 2129 Bal I 2 810, 864 BamH I 1 40Bbe I 2 1929, 2022 Bbs I 2 1761, 2071 Bcl I 1 1887 Bgl II 1 432 Bsa I 2 1715, 3723 BsaA I 3 1293, 2329, 4856 Bsp120 I 1 10 BspH I 2 3489, 4497 BspM I 1 1677 BsrG I 2 896, 2232 BssS I 3 2140, 2942, 4326 Bst98 I 2 1628, 1647 BstE II 1 2069 BstX I 2 506, 2431 BstZ I 1 468 Bsu36 I 1 46 Cfr10 I 4 2555, 2574, 3742, 4957

Enzyme # of Sites LocationCla I 1 476 Csp45 I 1 2639 Dde I 5 46, 3044, 3453, 3619,

4159 Dra I 3 3528, 3547, 4239 Dra III 2 2458, 4856 Drd I 3 1617, 2877, 4811 Dsa I 2 1313, 1878Eag I 1 468 Ear I 4 789, 797, 4457, 5145 EclHK I 1 3662 Eco52 I 1 468 Eco81 I 1 46 EcoICR I 2 495, 1527EcoR I 1 34 EcoR V 2 18, 1870 Ehe I 2 1927, 2020 Fsp I 2 3884, 5107Hinc II 4 30, 483, 1477, 2257 Hind II 4 30, 483, 1477, 2257 Hind III 2 1556, 2648 Hpa I 1 483 I-Ppo I 1 1651 Kas I 2 1925, 2018 Mlu I 1 502 Nae I 2 2557, 4959 Nar I 2 1926, 2019 Nco I 2 1313, 1878 Nde I 1 1187 NgoM IV 2 2555, 4957



Table 15. Restriction Enzymes That Cut the pGeneClip™ hMGFP Vector 1–5Times (continued).

Table 16. Restriction Enzymes that Do Not Cut the pGeneClip™ hMGFP Vector.

Table 17. Restriction Enzymes that Cut the pGeneClip™ hMGFP Vector 6 or MoreTimes.


Enzyme # of Sites LocationNhe I 1 1851 Not I 1 468 Nsi I 1 515 Nsp I 1 2773 PaeR7 I 1 487 PflM I 1 2458 PinA I 1 2574Ppu10 I 1 511 PspA I 1 1862 Pst I* 1 1638 Pvu I 4 1464, 1859, 4032,

5128 Pvu II 3 333, 729, 5157 Sac I 2 497, 1529 Sal I 1 28

Enzyme # of Sites LocationSca I 1 4142 Sgf I 2 1464, 1859 Sma I 1 1864 SnaB I 1 1293 Spe I 1 952 Ssp I 4 805, 852, 4466, 4648 Sty I 4 120, 1313, 1878, 2094 Tfi I 4 325, 740, 2175, 2744Tth111 I 1 2252 Vsp I 4 676, 735, 960, 3834 Xba I 2 22, 2568 Xho I 1 487 Xma I 1 1862 Xmn I 2 277, 4261

*A second Pst I site is created upon ligation of an insert.

Acc IIIAcc65 IAsc IAvr IIBbrP IBbu IBlp I

Bpu1102 IBsaB IBsaM IBsm IBssH IIBst1107 ICsp I

Dra IIEco47 IIIEco72 IEcoN IFse IKpn INru I

Pac IPme IPml IPpuM IPshA IPsp5 IIRsr II

Sac IISfi ISgrA ISph ISpl ISrf ISse8387 I

Stu ISwa IXcm I

Aci I Acy I Alu IAlw26 IAspH I Ava IIBan IBan IIBbv IBgl I BsaO I

BsaH I BsaJ I Bsp1286 I Bsr IBsrS IBst71 I BstO IBstU I Cfo IDpn I Dpn II

Eae I Fnu4H I Fok IHae IIHae III Hga I Hha I Hinf IHpa IIHph I Hsp92 I

Hsp92 IIMae I Mae IIMae IIIMbo I Mbo II Mnl I Mse I Msp IMspA1 INci I

Nde IINla IIINla IVPle I Rsa ISau3A ISau96 IScrF I SfaN ISin ITaq I

Tru9 IXho II


8.G . Composition of Buffers and Solutions

8.H. Related Products

Product Size Cat.#Pst I 3,000 units R6111

15,000 units R6115Pst I (HC) 15,000 units R4114

50,000 units R4117

RNA Production System

Product Size Cat.#T7 RiboMAX™ Express RNAi System 1 system P1700


LB plates with ampicillinAdd 15g agar to 1 liter of LB medium.Autoclave. Allow the medium to coolto 50°C before adding ampicillin to afinal concentration of 100µg/ml. Pour30–35ml of medium into 85mm petridishes. Let the agar harden. Store at4°C for up to 1 month or at roomtemperature for up to 1 week.

2M Mg2+ stock20.33g MgCl2 • 6H2O24.65g MgSO4 • 7H2O

Add distilled water to 100ml. Filtersterilize.

2X Rapid Ligation Buffer (provided)60mM Tris-HCl (pH 7.8)20mM MgCl2

20mM DTT2mM ATP10% polyethylene glycol

(MW8000, ACS Grade)Store in single-use aliquots at –20°C.Avoid multiple freeze-thaw cycles.

Oligo Annealing Buffer (provided)60mM Tris-HCl (pH 7.5)

500mM NaCl60mM MgCl2

10mM DTT

SOC medium (100ml)2.0g Bacto®-tryptone0.5g Bacto®-yeast extract1ml 1M NaCl

0.25ml 1M KCl1ml 2M Mg2+ stock, filter-

sterilized1ml 2M glucose, filter-sterilized

Add Bacto®-tryptone, Bacto®-yeastextract, NaCl and KCl to 97ml distilledwater. Stir to dissolve. Autoclave andcool to room temperature. Add 2MMg2+ stock and 2M glucose, each to afinal concentration of 20mM. Bring thevolume to 100ml with sterile, distilledwater. The final pH should be 7.0.

TE buffer10mM Tris-HCl (pH 8.0)1mM EDTA


RNAi Vector Systems

Product Size Cat.#psiCHECK™ 1 Vector 20µg C8011psiCHECK™ 2 Vector 20µg C8021

Reporter Assay Systems

Product Size Cat.#Renilla Luciferase Assay System 100 assays E2810

1,000 assays E2820Monster Green™ Fluorescent Protein phMGFP Vector 20µg E6421

Cell Proliferation Assay

Product Size Cat.#CellTiter-Glo® Luminescent Cell Viability Assay 10ml G7570

10 × 10ml G7571100ml G7572

10 × 100ml G7573

Plasmid DNA Purification

Product Size Cat.#Wizard® Plus SV Minipreps DNA Purification System 50 preps A1330

250 preps A1460Wizard® Plus SV Minipreps DNA Purification System + Vacuum Adapters 50 preps A1340

250 preps A1470




(a)This product is sold solely for use for research purposes in fields other than plants. This product is not transferable. If thepurchaser is not willing to accept the conditions of this label license, Promega is willing to accept the return of the productand provide the purchaser with a full refund. However if the product is used, then the purchaser agrees to be bound by theconditions of this limited use statement. This product is sold by Promega Corporation under license from Benitec AustraliaLtd and CSIRO as co-owners of U.S. Pat. No. 6,573,099 and foreign counterparts. For information regarding licenses to thesepatents for use of ddRNAi as a therapeutic agent or as a method to treat/prevent human disease, please contact Benitec [email protected]. For the use of ddRNAi in other fields, please contact CSIRO at: www.pi.csiro.au/RNAi. (b)This product is covered under license from Carnegie Institution of Washington under U.S. Pat. Nos. 6,506,559, 7,538,095,7,560,438, Australian Pat. No. 743798 and other patents pending. Commercial use of this product will require a separatelicense from Carnegie. (c)Patent Pending.(d)Certain applications of this product may require licenses from others.(e)BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USESTATEMENT. If the researcher is not willing to accept the conditions of this limited use statement, and the product is unused,Promega will accept return of the unused product and provide the researcher with a full refund.Researchers may use this product for research use only, no commercial use is allowed. Commercial Use means any and alluses of this product and derivatives by a party for monetary or other consideration and may include but is not limited to usein: (1) product manufacture; and (2) to provide a service, information or data; and/or resale of the product or its derivatives,whether or not such product or derivatives are resold for use in research. Researchers shall have no right to modify orotherwise create variations of the nucleotide sequence of the Monster Green® gene except that researchers may create fusedgene sequences provided that the coding sequence of the resulting Monster Green® gene has no more than fourdeoxynucleotides missing at the affected terminus compared to the intact Monster Green® gene sequence. No other use ortransfer of this product or derivatives is authorized without the prior express, written consent of Promega Corporation.Researchers may transfer derivatives to others for research use provided that at the time of transfer a copy of this label licenseis given to the recipients and recipients agree to be bound by the terms of this label license. PROMEGA MAKES NOREPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FORMERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THE PRODUCT. The terms ofthis agreement shall be governed under the laws of the State of Wisconsin, USA. (f)Australian Pat. No. 2003297293 and other patents pending.(g) The gene encoding Monster Green® Fluorescent Protein is exclusively licensed to Promega Corporation under U.S. Pat.Nos. 7,291,711 and 7,413,874 and other patents pending.

© 2011 Promega Corporation. All Rights Reserved.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for moreinformation.CellTiter-Glo and Wizard are registered trademarks of Promega Corporation. Monster Green, GeneClip, psiCHECK andRiboMAX are trademarks of Promega Corporation.Bacto is a registered trademark of Difco Laboratories, Detroit, Michigan. DH5α is a trademark of Life Technologies, Inc.DNASTAR is a registered trademark of DNASTAR, Inc. GenBank is a registered trademark of US Dept of Health and HumanServices.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for moreinformation.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for themost up-to-date information on Promega products.


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GeneClip™ U1 Hairpin Cloning Systems