QuikChange™ XL Site-Directed Mutagenesis...

QuikChange™ XL Site-DirectedMutagenesis Kit

INSTRUCTION MANUALCatalog #200516Revision #100002

For in Vitro Use Only

*200516-12__100002*/

LIMITED PRODUCT WARRANTYThis warranty limits our liability to replacement of this product. No other warranties of any kind,express or implied, including without limitation, implied warranties of merchantability or fitness for aparticular purpose, are provided by Stratagene. Stratagene shall have no liability for any direct,indirect, consequential, or incidental damages arising out of the use, the results of use, or the inabilityto use this product.

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QuikChange™ XL Site-Directed Mutagenesis Kit

CONTENTSMaterials Provided....................................................................................................................... 1Storage Conditions ....................................................................................................................... 1Additional Materials Required .................................................................................................... 1Introduction.................................................................................................................................. 2Primer Design............................................................................................................................... 5XL10-Gold Ultracompetent Cells ................................................................................................ 6Protocol......................................................................................................................................... 7

QuikSolution................................................................................................................... 7Setting Up the Reactions ................................................................................................. 7Cycling the Reactions...................................................................................................... 9Digesting the Products..................................................................................................... 9

Transformation Guidelines ..........................................................................................................10Storage Conditions ..........................................................................................................10Aliquoting Cells ..............................................................................................................10Use of Falcon 2059 Polypropylene Tubes........................................................................10Use of �-Mercaptoethanol ...............................................................................................10Quantity of DNA Added..................................................................................................10Length and Temperature of the Heat Pulse.......................................................................10Preparing the Agar Plates for Color Screening .................................................................10Transforming XL10-Gold Ultracompetent Cells ..............................................................11

Troubleshooting............................................................................................................................13Preparation of Media and Reagents ............................................................................................15References.....................................................................................................................................16Endnotes .......................................................................................................................................16Quick-Reference Protocol ............................................................................................................19

QuikChange™ XL Site-Directed Mutagenesis Kit 1

QuikChange™ XL Site-Directed Mutagenesis Kit

MATERIALS PROVIDEDMaterial provideda QuantityPfuTurbo® DNA polymerase (2.5 U/ �l) 30 reactions10× reaction bufferb 1 mlDpn I restriction enzyme (10 U/�l) 300 UOligonucleotide control primer #1 [34-mer (100 ng/�l)]

5´ CCA TGA TTA CGC CAA GCG CGC AAT TAA CCC TCA C 3´750 ng

Oligonucleotide control primer #2 [34-mer (100 ng/�l)]5´ GTG AGG GTT AAT TGC GCG CTT GGC GTA ATC ATG G 3´

750 ng

pWhitescript™ 4.5-kb control plasmid (5 ng/ �l) 50 ngQuikSolution 500 �ldNTP mixc 30 �lXL10-Gold ultracompetent cellsd 10 × 135 �lXL10-Gold �-mercaptoethanol mix (�-ME) 2 × 50 �lpUC18 control plasmid (0.1 ng/�l in TE bufferb) 10 �l

a The QuikChange XL site-directed mutagenesis kit contains enough reagents for 30 reactions (25test reactions and 5

control reactions).b See Preparation of Media and Reagents.c Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do notDo notDo notDo notsubject the dNTP mixsubject the dNTP mixsubject the dNTP mixsubject the dNTP mix

to multiple freeze-thaw cycles.to multiple freeze-thaw cycles.to multiple freeze-thaw cycles.to multiple freeze-thaw cycles.d Genotype: Tetr� (mcrA)183 �(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lacHte

[F’ proAB lacIqZ�M15 Tn10 (Tetr) Amy Camr]

STORAGE CONDITIONSXL10-Gold Ultracompetent cells, XL10-Gold ��-ME, and pUC18 Control Plasmid: –80°CAll Other Components: –20°C

ADDITIONAL MATERIALS REQUIREDFalcon® 2059 polypropylene tubes (15 ml)5-Bromo-4-chloro-3-indoyl-�-D-galactopyranoside (X-gal)Isopropyl-1-thio-�-D-galactopyranoside (IPTG)

Revision #100002 Copyright © 2000 by Stratagene.

2 QuikChange™ XL Site-Directed Mutagenesis Kit

INTRODUCTIONIn vitro site-directed mutagenesis is an invaluable technique for studyingprotein structure-function relationships and gene expression, and forcarrying out vector modification. Several approaches to this technique havebeen published, but these methods generally require single-stranded DNA(ssDNA) as the template1–4 and are labor intensive or technically difficult.Stratagene’s QuikChange™ XL site-directed mutagenesis kit* allows site-specific mutation in virtually any double-stranded plasmid, thus eliminatingthe need for subcloning into M13-based bacteriophage vectors and forssDNA rescue.5 In addition, the QuikChange XL system requires nospecialized vectors, unique restriction sites, or multiple transformations.This rapid four-step procedure generates mutants with greater than 80%efficiency. The protocol is simple and uses either miniprep plasmid DNA orcesium-chloride-purified DNA.

The QuikChange XL system is used to make point mutations, switch aminoacids, and delete or insert single or multiple amino acids. The QuikChangeXL method is performed using PfuTurbo® DNA polymerasell,** and athermal temperature cycler. PfuTurbo DNA polymerase replicates bothplasmid strands with high fidelity and without displacing the mutantoligonucleotide primers. The basic procedure utilizes a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two syntheticoligonucleotide primers containing the desired mutation (see Figure 1). Theoligonucleotide primers, each complementary to opposite strands of thevector, are extended during temperature cycling by using PfuTurbo DNApolymerase. Incorporation of the oligonucleotide primers generates amutated plasmid containing staggered nicks. Following temperature cycling,the product is treated with Dpn I. The Dpn I endonuclease (target sequence:5 -́Gm6ATC-3 )́ is specific for methylated and hemimethylated DNA and isused to digest the parental DNA template and to select for mutation-containing synthesized DNA.7 DNA isolated from almost all Escherichiacoli strains is dam methylated‡ and therefore susceptible to Dpn I digestion.The nicked vector DNA incorporating the desired mutations is thentransformed into XL10-Gold®*** ultracompetent cells. The small amount ofstarting DNA template required to perform this method, the high fidelity ofthe PfuTurbo DNA polymerase, and the low number of thermal cycles allcontribute to the high mutation efficiency and decreased potential forrandom mutations during the reaction.

* U.S. Patent Nos. 5,789,166 and 5,923,419 and Patents Pending.ll U.S. Patent No. 5,545,552 and 5,866,395 and 5,948,663 and Patents Pending** PfuTurbo DNA polymerase has 6-fold higher fidelity in DNA synthesis than Taq DNA

polymerase.‡ Ensure that the plasmid DNA to be used as starting material in the mutagenesis reaction

is isolated from a dam+ E. coli strain. Plasmid DNA isolated from dam– strains (JM110or SCS110, for example) is not suitable.

***U.S. Patent Nos. 5,512,486 and 5,707,841 and Patents Pending.


FIGURE 1 Overview of the QuikChange™ XL site-directed mutagenesis method.


The QuikChange XL site-directed mutagenesis kit described in this manualis a new version of Stratagene's popular QuikChange site-directedmutagenesis kit. The QuikChange XL kit was created for efficientmutagenesis of large or otherwise difficult-to-mutagenize plasmidtemplates. The QuikChange XL kit features components specificallydesigned for more efficient DNA replication and bacterial transformation.The QuikChange solution is provided to facilitate replication of largeplasmids, while XL10-Gold ultra competent cells have been included toensure the highest transformation efficiencies possible. The transformationefficiency of XL10-Gold cells is 5-fold higher than the transformationefficiency of XL1-Blue cells employed in the standard QuikChange kit8.Moreover, XL10-Gold cells contain the Hte phenotype, which increases thetransformation efficiency of larger DNA plasmids.

To demonstrate the effectiveness of the QuikChange XL method, thepWhitescript™ 4.5-kb control plasmid is used to test the efficiency ofmutant plasmid generation. The pWhitescript 4.5-kb control plasmidcontains a stop codon (TAA) at the position where a glutamine codon(CAA) would normally appear in the �-galactosidase gene of thepBluescript® II SK(–) phagemid (corresponding to amino acid 9 of theprotein). XL10-Gold ultracompetent cells transformed with this controlplasmid appear white on LB–ampicillin agar plates (see Preparation ofMedia and Reagents), containing IPTG and X-gal, because �-galactosidaseactivity has been obliterated. The oligonucleotide control primers create apoint mutation that reverts the T residue of the stop codon (TAA) in the�-galactosidase gene encoded on the pWhitescript 4.5-kb control template tothe C residue to produce a glutamine codon (Gln, CAA). Followingtransformation, colonies can be screened for the �-galactosidase (�-gal+)(blue) phenotype.


PRIMER DESIGN

Note Stratagene offers Custom Oligonucleotide Synthesis, a servicewhich synthesizes custom oligodeoxynucleotides to meet specificresearch needs. Polyacrylamide gel electrophoresis (PAGE)purification and 5´ phosphorylation are also available. Pleasecontact our Technical Services Department at (800) 894-1304 oryour local distributor for additional information or fax a typed 5´to 3´ sequence to (512) 321-3202 (Attention: Custom OligoDepartment).

The mutagenic oligonucleotide primers for use in this protocol must bedesigned individually according to the desired mutation. The followingconsiderations should be made for designing mutagenic primers:

1. Both mutagenic primers must contain the desired mutation and annealto the same sequence on opposite strands of the plasmid.

2. Primers should be between 25 and 45 bases in length, and the meltingtemperature (Tm) of the primers should be greater than or equal to78°C. The following formula is commonly used for estimating the Tmof primers:

mismatch % 675/ 0.41(%GC) + 81.5 m �� NT

For calculating Tm:� N is the primer length in bases.� values for %GC and % mismatch are whole numbers

For calculating Tm for primers intended to introduce insertions ordeletions, use this modified version of the above formula:

NT 675/ 0.41(%GC) + 81.5 m ��

where N does not include the bases which are being inserted or deleted

3. The desired mutation (deletion or insertion) should be in the middle ofthe primer with ~10–15 bases of correct sequence on both sides.

4. The primers optimally should have a minimum GC content of 40% andshould terminate in one or more C or G bases.

5. Primers need not be 5 ́phosphorylated but must be purified either byfast polynucleotide liquid chromatography (FPLC) or bypolyacrylamide gel electrophoresis (PAGE). Failure to purify theprimers results in a significant decrease in mutation efficiency.

6. It is important to keep primer concentration in excess. Stratagenesuggests varying the amount of template while keeping theconcentration of the primer constantly in excess.


XL10-GOLD ULTRACOMPETENT CELLSStratagene's XL10-Gold ultracompetent cells, a derivative of Stratagene'shighest-efficiency competent cell line XL2-Blue MRF ,́ contain the Htephenotype which increases transformation efficiency of ligated DNA.8XL10-Gold cells are both endonuclease deficient (endA1) andrecombination deficient (recA). The endA1 mutation greatly improves thequality of plasmid miniprep DNA, and the recA mutation helps ensure insertstability. In addition, the McrA, McrCB, McrF, Mrr, and HsdR systems havebeen removed from XL10-Gold ultracompetent cells. The mcrA, mcrCB andmrr mutations prevent cleavage of cloned DNA that carries cytosine and/oradenine methylation, which is often present in eukaryotic DNA andcDNA.9–13 The McrA and McrCB systems recognize and restrict methylatedcytosine DNA sequences, and the Mrr system recognizes and restrictsmethylated adenine DNA sequences. The Mrr system also restrictsmethylated cytosine DNA sequences with a specificity differing from that ofMcrA and McrCB. This activity has been named McrF. This McrF activityagainst methylated cytosines has been shown to be equal to or greater thanthe restriction activity of the McrA and McrCB systems.14 The hsdRmutation prevents the cleavage of cloned DNA by the EcoK (hsdR)endonuclease system. XL10-Gold cells grow faster than XL1 or XL2-Bluecells, resulting in larger colonies. To permit blue-white color screening, theXL10-Gold ultracompetent cells contain the lacIqZ�M15 gene on theF ́episome.

Host strain References GenotypeXL10-Goldultracompetentcells

8, 15, 16 TetR �(mcrA)183 �(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1gyrA96 relA1 lac Hte [F´ proABlacIqZ�M15 Tn10 (TetR) Amy CamR]a

a Chloramphenicol resistant (CamR) at concentrations of <40 �g/ml, butchloramphenicol sensitive (CamS) at concentrations of 100 �g/ml.

It is important to store the XL10-Gold supercompetent cells at –80°C toprevent a loss of efficiency. For best results, please follow the directionsoutlined in the following sections.7


PROTOCOL

QuikSolutionQuikSolution has been shown to improve linear amplification. Enhancedamplification efficiencies are observed when using between 2.5–3.5 �lQuikSolution/50 �l reaction, with 3 �l being optimal for most targets.

Setting Up the Reactions

Note To maximize temperature-cycling performance, Stratagenestrongly recommends using Thin-Wall Tubes, which are optimizedand tested to ensure ideal contact with the temperature cycler’sheat blocks.

1. Synthesize two complimentary oligonucleotides containing the desiredmutation, flanked by unmodified nucleotide sequence. Purify theseoligonucleotide "primers" prior to use in the following steps (seePrimer Design).

2. Prepare the control reaction as indicated below:

5 �l of 10× reaction buffer 2 �l (10 ng) of pWhitescript™ 4.5-kb control plasmid (5 ng/�l) 1.25 �l (125 ng) of oligonucleotide control primer #1

[34-mer (100 ng/�l)] 1.25 �l (125 ng) of oligonucleotide control primer #2

[34-mer (100 ng/�l)] 1 �l of dNTP mix 3 �l QuikSolution36.5 �l Double-distilled water (ddH2O) to a final volume of 50 �l

Then add

1 �l of PfuTurbo DNA polymerase (2.5 U/�l)


3. Prepare the sample reaction(s) as indicated below:

Note Stratagene recommends setting up an initial sample reactionusing 10 ng of dsDNA template. If this initial reaction isunsuccessful, set up a series of sample reactions usingvarious concentrations of dsDNA template ranging from 5 to50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA template) whilekeeping the primer concentration constant.

5 �l of 10× reaction bufferX �l (10 ng) of dsDNA templateX �l (125 ng) of oligonucleotide primer #1X �l (125 ng) of oligonucleotide primer #21 �l of dNTP mix3 �l QuikSolutionddH2O to a final volume of 50 �l

Then add

1 �l of PfuTurbo DNA polymerase (2.5 U/�l)

4. If the thermal cycler to be used does not have a hot top assembly,overlay each reaction with 30 �l of mineral oil.

TABLE I

Cycling Parameters for the QuikChange XL MethodSegment Cycles Temperature Time1 1 95°C 1 minute

95°C 50 seconds60°C 50 seconds

2 18

68°C 2 minutes/kb of plasmidlength

3 1 68°C 7 minutes


Cycling the Reactions1. Cycle each reaction using the cycling parameters outlined in Table I.

Note It is important to adhere to the 18-cycle limit when cyclingthe mutagenesis reactions. More that 18 cycles can havedeleterious effects on the reaction efficiency.

2. Following temperature cycling, place the reaction on ice for 2 minutesto cool the reaction to �37°C.

Note If desired, amplification may be checked by electrophoresis of10 µl of the product on a 1% agarose gel. A band may or may notbe visualized at this stage. In either case proceed with Dpn Idigestion and transformation.

Digesting the Products

Note It is important to insert the pipet tip below the mineral oil overlay(if used) when adding the Dpn I restriction enzyme to the reactiontubes during the digestion step or when transferring the 1 �l of theDpn I-treated DNA required for the transformation reaction.Stratagene suggests using specialized aerosol-resistant pipet tips,which are small and pointed, to facilitate this process.

1. Add 1 �l of the Dpn I restriction enzyme (10 U/�l) directly to eachamplification reaction below the mineral oil overlay using a small,pointed pipet tip.

2. Gently and thoroughly mix each reaction mixture by pipetting thesolution up and down several times. Spin down the reaction mixtures ina microcentrifuge for 1 minute and immediately incubate each reactionat 37°C for 1 hour to digest the parental (i.e., the nonmutated)supercoiled dsDNA.


TRANSFORMATION GUIDELINES

Storage ConditionsUltracompetent cells are sensitive to even small variations in temperatureand must be stored at the bottom of a –80°C freezer. Transferring tubes fromone freezer to another may result in a loss of efficiency. Ultracompetentcells should be placed at –80°C directly from the dry ice shipping container.

Aliquoting CellsWhen aliquoting, keep ultracompetent cells on ice at all times. It is essentialthat the Falcon® 2059 polypropylene tubes are placed on ice before the cellsare thawed and that the cells are aliquoted directly into the prechilled tubes.

Use of Falcon 2059 Polypropylene TubesIt is important that Falcon® 2059 polypropylene tubes are used for thetransformation protocol, since other tubes may be degraded by the�-mercaptoethanol used in step 3 of the Transformation Protocol. Inaddition, the incubation period during the heat-pulse step is critical and hasbeen calculated specifically for the thickness and shape of the Falcon 2059polypropylene tubes.

Use of �-Mercaptoethanol�-Mercaptoethanol (�-ME) has been shown to increase transformationefficiency. The XL10-Gold �-mercaptoethanol mix provided in this kit isdiluted and ready to use.

Quantity of DNA AddedGreatest efficiencies are observed when adding 2 �l of the ligation mixture.A greater number of colonies will be obtained when adding up to 50 ng,although the overall efficiency may be lower.

Length and Temperature of the Heat PulseThere is a defined window of highest efficiency resulting from the heatpulse during transformation. Optimal efficiencies are observed when cellsare heat-pulsed for 30 seconds. Heat-pulsing for at least 30 seconds isrecommended to allow for slight variations in the length of incubation.Efficiencies decrease when incubating for <30 seconds or for >40 seconds.Do not exceed 42°C.

Preparing the Agar Plates for Color ScreeningTo prepare the LB agar plates for blue–white color screening, add 80 �g/mlof 5-bromo-4-chloro-3-inodlyl-�-D-galactopyranisode (X-gal), 20 mMisopropyl-1-thio-�-D-galactopyrano-side (IPTG), and the appropriateantibiotic to the LB agar. Alternatively, 100 �l of 10 mM IPTG and 100 �lof 2% X-gal can be spread on the LB agar plates 30 minutes prior to platingthe transformations. Prepare the IPTG in sterile dH2O; prepare the X-gal indimethylformamide (DMF).

Some �-galactosidase fusion proteins are toxic to the host bacteria. If aninsert is suspected to be toxic to the bacteria, do not plate the


transformations with X-gal and IPTG. Color screening will be eliminated,but the recombinants will express lower levels of potentially toxic proteins.

Transforming XL10-Gold Ultracompetent Cells

Note Please read the Transformation Guidelines before proceeding withthe transformation protocol.

1. Gently thaw the XL10-Gold ultracompetent cells on ice. For eachcontrol and sample reaction to be transformed, aliquot 45 �l of thesupercompetent cells to a prechilled Falcon 2059 polypropylene tube.

2. Add 2 �l of the �-ME mix provided with the kit to the 45 �l ofcells. (Stratagene cannot guarantee highest efficiencies with �-ME fromother sources.)

3. Swirl the contents of the tube gently. Incubate the cells on ice for10 minutes, swirling gently every 2 minutes.

4. Transfer 2 �l of the Dpn I-treated DNA from each control and samplereaction to separate aliquots of the supercompetent cells.

Note Carefully remove any residual mineral oil from the pipet tipbefore transferring the Dpn I-treated DNA to each reaction.

Swirl the transformation reactions gently to mix and incubate thereactions on ice for 30 minutes.

As an optional step, verify the transformation efficiency of theXL10-Gold ultracompetent cells by adding 1 �l of 0.1 ng/�l pUC18control plasmid (dilute the control provided 1:10 in high-quality water)to a 45-�l aliquot of the supercompetent cells and incubating asindicated above.

5. Preheat NZY+ broth (see Preparation of Media and Reagents) in a42°C water bath for use in step 8.

Note Transformation of XL10-Gold ultracompetent cells has beenoptimized for use with NZY+ broth.

6. Heat-pulse the tubes in a 42°C water bath for 30 seconds. The durationof the heat pulse is critical for obtaining the highest efficiencies. Do notexceed 42°C.

Note This heat pulse has been optimized for the Falcon 2059polypropylene tubes.

7. Incubate the tubes on ice for 2 minutes.

8. Add 0.5 ml of preheated (42°C) NZY+ broth to each tube and incubatethe tubes at 37°C for 1 hour with shaking at 225–250 rpm.

9. Plate the transformation reactions as follows:


a. Plate 250 �l of the pWS 4.5 control transformation reaction andonly 5 �l of the pUC18 control transformation reaction (ifperformed) on LB–ampicillin agar plates that have been preparedwith 20 �l of 10% (w/v) X-gal and 20 �l of 100 mM IPTG (seePreparing the Agar Plates for Color Screening).

Note When plating the pUC18 control transformationreaction, first pipet a 200-�l pool of NZY+ broth ontothe plate and add the 5 �l of pUC18 controltransformation to the pool. Spread the mixture with asterile spreader.

b. Plate the entire volume of each sample transformation reactionon agar plates containing the appropriate antibiotic for theplasmid vector.

10. Incubate the transformation plates at 37°C for >16 hours.

Expected Results for the Control TransformationsThe expected colony number from the pWS 4.5 control transformationsshould be between 50 and 800 colonies. Greater than 80% of themutagenized control colonies should contain the mutation and appear asblue colonies on agar plates containing IPTG and X-gal.

Note The mutagenesis efficiency (ME) for the pWhitescript 4.5-kbcontrol plasmid is calculated by the following formula:

ME Number of blue colony forming units (cfu)Total number of colony forming units (cfu)

� � 100%

If transformation of the pUC18 control plasmid was performed,>100 colonies (>109 cfu/�g) should be observed, with >98% having theblue phenotype.

Expected Results for Sample TransformationsThe expected colony number should be between 10 and 1000 colonies,depending upon the base composition and length of the DNA templateemployed. For suggestions on increasing colony number, seeTroubleshooting. The insert of interest should be sequenced to verify thatselected clones contain the desired mutation(s).


TROUBLESHOOTINGWhen used according to the guidelines outlined in this instruction manual, Stratagene’s kit will providea reliable means to conduct site-directed mutagenesis using dsDNA templates. Undoubtedly, there willbe variations in the base composition and length of the DNA template and in the thermal cycler that maycontribute to differences in mutagenesis efficiency. Stratagene provides the following guidelines fortroubleshooting these variations.

Observation Suggestion(s)Ensure that sufficient DNA template is used in the reaction. Visualize the DNAtemplate on a gel to verify the quantity and quality. Repeat reaction using higheramounts of plasmid DNA (100 ng, 200 ng, 500 ng)Ensure that sufficient mutant DNA is synthesized in the reaction.� Titrate QuikSolution in 1-�l increments from 0 to 5 �l� Increase the amount of the Dpn I-treated DNA used in the transformation

reaction to 4 �l� Increase the extension time to 2.5 min/kb� Precipitate entire reaction and use all of it in the transformation

Ensure sufficient mutant DNA is synthesized by adjusting the cycling parameters forthe sample reaction to overcome differences in ramping efficiencies of thermalcyclers. Increase initial denaturation step (segment 1) to 1–2 minutes anddenaturation cycles (segment 2) to 1 minute

Lowtransformationefficiency or lowcolony number

Ensure that excess mineral oil is not transferred into the transformation reactionwhen pipetting the Dpn I-treated DNA. Using the smallest pipet tips available, insertthe pipet tip completely below the mineral layer overlay and clear the pipet tip whilesubmerged beneath the mineral oil overlay before collecting the sampleDifferent thermal cyclers contribute to variations in cycling efficiencies. Adjust thecycling parameters (including ramp rates) for the control reaction and repeat theprotocol for the sample reactionsEnsure that supercompetent cells are stored at the bottom of a –80°C freezerimmediately upon arrival; use XL10-Gold �-ME in the transformation reactions (seealso Transformation Guidelines)Verify that the agar plates were prepared correctly. To prepare the LB–ampicillin agarplates for the transformed control cells, pipet 20 �l of 10% (w/v) X-gal (prepared inDMF) and 20 �l of 100 mM IPTG (prepared in filter-sterilized dH2O) into a 100-�lpool of NZY+ broth and spread the mixture across the plate (see Preparing the AgarPlates for Color Screening)For best visualization of the blue (�-gal+) phenotype, the control plates must beincubated for at least 16 hours at 37°C

Lowmutagenesisefficiency or lowcolony numberwith the controlreaction

Avoid multiple freeze-thaw cycles for the dNTP mix. Thaw the dNTP mix once,prepare single-use aliquots, and store the aliquots at –20°C. Do not subject thedNTP mix to multiple freeze-thaw cyclesAdd the Dpn I restriction enzyme below the mineral oil overlay in the digestion stepand ensure proper mixing of all components in the reaction especially the Dpn I

Lowmutagenesisefficiency withthe samplereaction(s)

Allow sufficient time for the Dpn I to completely digest the parental template; repeatthe digestion if too much DNA template was present. Increase digestion time to 1.5–2.0 hours


Avoid multiple freeze-thaw cycles for the dNTP mix. Thaw the dNTP mix once,prepare single-use aliquots, and store the aliquots at –20°C. Do not subject thedNTP mix to multiple freeze-thaw cyclesPoor quality primers can lead to false positives. Radiolabel the primers and check fordegradation on an acrylamide gel or resynthesize the primers

False positives

False priming can lead to false positives. Increase the stringency of the reaction byincreasing the annealing temperature to within 5°C of the melting temperature ofthe mutation primers


PREPARATION OF MEDIA AND REAGENTS

LB Agar (per Liter)10 g of NaCl10 g of tryptone 5 g of yeast extract20 g of agarAdd deionized H2O to a final volume of

1 literAdjust pH to 7.0 with 5 N NaOHAutoclavePour into petri dishes (~25 ml/100-mm

plate)

LB–Ampicillin Agar (per Liter)(Use for reduced satellite colony formation)1 liter of LB agarAutoclaveCool to 55°CAdd 50 mg of filter-sterilized ampicillinPour into petri dishes (~25 ml/100-mm

plate)

10× Reaction Buffer100 mM KCl100 mM(NH4)2SO4200 mM Tris-HCl (pH 8.8) 20 mM MgSO41% Triton® X-100��mg/ml nuclease-free bovine serum

albumin (BSA)

NZY+ Broth (per Liter)10 g of NZ amine (casein hydrolysate) 5 g of yeast extract 5 g of NaClAdjust to pH 7.5 using NaOHAutoclaveAdd the following supplement prior to use

12.5 ml of 1 M MgCl2 and 12.5 ml of 1 M MgSO4

10 ml of a 2 M filter-sterilized glucose solution or 20 ml of 20% (w/v) glucose

Filter sterilize

TE Buffer10 mM Tris-HCl (pH 7.5) 1 mM EDTA


REFERENCES 1. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 488. 2. Vandeyar, M., Weiner, M. P., Hutton, C., and Batt, C. (1988) Gene 65: 129–133. 3. Sugimoto, M., Esaki, N., Tanaka, H., and Soda, K. (1989) Anal. Biochem.

179: 309–311. 4. Taylor, J. W., and Eckstein, F. (1985) Nucleic Acids Res. 13: 8764. 5. Papworth, C., Braman, J., and Wright, D. A. (1996) Strategies 9(1): 3–4. 6. Bergseid, M., Scott, B., Mathur, S., Nielson, K., Shoemaker, D., and Mathur, E. (1991)

Strategies 4(3): 34–35. 7. Nelson, M., and McClelland, M. (1992) Methods Enzymol. 216: 279–303. 8. Jerpseth, A., et al. (1997) Strategies: 10: 37-38. 9. Hanahan, D. (1983) J. Mol. Biol. 166: 557–580.10. Yanish-Peron, C., Viera, J., and Messing, J. (1985) Gene 33: 103–199.11. Hatt, J., Callahan, M., and Greener, A. (1992) Strategies 5: 2–3.12. Kelleher, J. E., and Raleigh, E. A. (1991) J. Bacteriol. 173: 5220–5223.13. Kohler, S. W., et al. (1990) Nucleic Acids Res. 18: 3007–3013.14. Gough, J., and Murray, N. (1983) J. Mol. Biol. 166: 1–19.15. Bullock, W. O., Fernandez, J. M., and Short, J. M. (1987) Biotechniques 5: 376–379.16. Greener, A., and Jerpseth, B. (1993) Strategies 6(2): 57.

ENDNOTESpBluescript®and PfuTurbo® are registered trademarks of Stratagene in the United States.pWhitescript and QuikChange are trademarks of Stratagene.Falcon® is a registered trademark of Becton Dickinson and Company.Triton® is a registered trademark of Rohm and Haas Co.

19

QuikChange™ XL Site-Directed Mutagenesis KitCatalog #200516

QUICK-REFERENCE PROTOCOL� Prepare the control and sample reaction(s) as indicated below:

Note Stratagene recommends setting up an initial sample reaction using 10 ng of dsDNAtemplate. If this initial sample reaction is unsuccessful, set up a series of reactionsusing various concentrations of dsDNA template ranging from 5 to 50 ng (e.g., 5, 10,20, and 50 ng of dsDNA template) while keeping the primer concentration constant.

Control Reaction 5 �l of 10× reaction buffer 2 �l (10 ng) of pWhitescript™ 4.5-kb

control template (5 ng/�l) 1.25 �l (125 ng) of oligonucleotide

control primer #1 [34-mer (100ng/�l)]

1.25 �l (125 ng) of oligonucleotidecontrol primer #2 [34-mer (100ng/�l)]

1 �l of dNTP mix 3 �l of QuikSolution36.5 �l ddH2O to a final volume of 50 �l

Sample Reaction5 �l of 10× reaction bufferX �l (10 ng) of dsDNA templateX �l (125 ng) of oligonucleotide primer

#1X �l (125 ng) of oligonucleotide primer

#21 �l of dNTP mix3 �l of QuikSolutionddH2O to a final volume of 50 �l

� Then add 1 �l of PfuTurbo DNA polymerase (2.5 U/�l) to each control andsample reaction

� If the thermal cycler to be used does not have a hot top assembly, overlay eachreaction with 30 �l of mineral oil.

� Cycle each reaction using the cycling parameters outlined in the followingtable:

Segment Cycles Temperature Time1 1 95°C 1 minute

95°C 50 seconds60°C 50 seconds

2 18

68°C 2 minutes/kb of plasmidlength

3 1 68°C 7 minutes

� Add 1 �l of the Dpn I restriction enzyme (10 U/�l) below the mineral oil overlay

20

� Gently and thoroughly mix each reaction, spin down in a microcentrifuge for 1minute, and immediately incubate at 37°C for 1 hour to digest the parentalsupercoiled dsDNA

� Transform 2 �l of the Dpn I-treated DNA from each control and samplereaction into separate 45-�l aliquots of XL10-Gold ultracompetent cells (seeTransforming XL10-Gold Ultracompetent Cells in the instruction manual)

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