Version Sept. 2014.

Gelb Lab Site-Directed Mutagenesis Manual

Introduction. We have had good success making mutants using the QuikChange kit from Stratagene or the Sculptor kit from Amersham. But, starting end of 2013 we have been using the mutagenesis kit from NEB and it works great. We follow the kit instructions exactly. I suggest you try this kit as the first choice method. The kit is NEB Cat. E0554S Q5 site directed mutagenesis kit, buy it from UW Biochem. Strockroom to save shipping costs and get the UW discount.

Below we give the details for the QuickChange and Sculptor kits (our old protocols), but again try the NEB kit first.

 Info for QuickChange and Sculptor Kits.

QuikChange has worked in most cases and should be tried first as it is the easiest and fastest to execute. One exception is for big plasmids like pVL for Baculovirus or some mammalian expression vectors. If the plasmid + insert is much above ~6000 nt you may want to put your insert into a small vector such as a pUC vector, make the mutant using QuikChange and then transfer the insert back to your desired plasmid. Of you can try the new modified QuickChange kit from Stratagene that is optimized for large plasmids. If QuikChange does not work, you can try the Sculptor kit. QuikChange starts with a double stranded plasmid, whereas Sculptor requires the preparation of a single stranded plasmid template, thus the QuikChange approach is faster. It is the policy of the Gelb lab, that the entire coding region of all mutated genes must be fully sequenced to verify not only that the mutation was introduced, but that there are no other mutations in the coding region. Finally we no longer buy the QuikChange Kit from Stratagene since it is much cheaper to buy the components separately and this works just fine.

The Pfu Turbo DNA polymerase is purchased from Stratagene. DpnI is from New England Biolabs or from the UW Biochemistry stockroom. The 10 mM dNTP stock is made from ATP,GTP,CTP, and TTP purchased at the UW Biochemistry Stock Room, the final concentration of nucleotide triphosphophates is 0.2 mM in the PCR reaction. If you want to do a positive control to be sure the mutagenesis is working, we have a stock stolution of the same beta-galactosidase plasmid that comes with the QuickChange kit (pWhitescript plasmid) and we also have the set of oligos needed to mutate this beta-galactosidase. For mutagenesis, follow the QuickChange manual and use the same concentration of components that Stratagene recommends. You should use XL1-Blue with electroporation for the transformation since not all E.coli strains have the capacity to repair the nicked plasmids generated from the QuickChange procedure.

Stragene Quik Change Manual

Design of oligos for mutagenesis. Two oligos are needed for QuickChange, whereas one is needed for Sculptor. We use the oligos as supplied by the company without further purification (despite the fact that Stratagene suggests gel purification of oligos for QuickChange, we have found this not to be necessary). For Sculptor but not for QuickChange, the oligo has to be 5'-phosphorylated.

For any kind of mutagenesis, you should check your oligos for stemloop formation and duplex formation with itself. To do this use the OLIGO program that runs on the PC in the lab. You simply type in the sequence of your oligo and it shows you any stemloop formation and any self-duplex formation. Avoid oligos that have extensive stemloop and/or self-duplex formation.

Oligos for OuickChange. The oligos should be ~30 nt in length (longer for multi-site mutations, see below). The 3' nucleotide should be a G or a C. The mutation region should be in the center of the oligo. The minimum GC content of the oligo is 40% (we have successfully used 40-75%). To make a single point mutation, try, as a first choice, to change only one nucleotide. If the genetic code does not permit this, we have been able to change 2-3 nucleotides in the codon with a single oligo. We have been able to make up to 3 amino acid changes with a single oligo. For this, the oligo should have 9-10 perfect match nucleotides at each end of the oligo. Here are two examples.

To make the R7E/K1OE/K16E mutant of human group IIa PLA2, we used the oligo:

GAA TTT CCA CGA GAT GAT CGA GTT GAC GAC AGG AGA GGA AGC CGC AC

The mutation sites are underscored. This is a 47-mer, with 10 nucleotides of perfect match before the first mutation site and 12 after the last mutation site.

For QuickChange, you also need to make the oligo that is the exact complement of the above oligos (i.e. not the wild type sequence but bearing the desired mutations).

Oligos for Sculptor. Use the same rules as for QuickChange oligos except for single-amino acid mutations, the oligo should be ~20 bp instead of 30 bp.

An example is the oligo to make the K124E/R127D mutant of human group IIa PLA2, we used the oligo:

CGG GGT CGA CCC ATC GCA GTG TTC GTT AGA GTA G

This oligo is a 34-mer with 13 nucleotides of perfect match before the first mutation site and 10 after the last mutation.

QuickChange Mutagenesis. We follow the exact procedure from Stratagene that comes with the kit except for the following change. We do the mutagenesis reaction at half the scale that the company gives (this saves reagents). Stratagene recommends using 5-50 ng of template DNA, but we always use 25 ng template DNA for half-size reaction (quantified using fluorescence dye binding assay, see Gelb lab handout). We use 62.5 ng of each oligo for half-size reaction. The oligos typically come in lyophilized form. We hydrate them with 200 ml TE buffer and store them at -20° C. An aliquot of this stock is used to quantify the amount of DNA by the OD260 (OD260 = 1, in a 1 cm path-length cuvete in a volume of 1 ml is 33 mg of DNA). A portion of the oligo stock solution is further diluted with water to give 50 ng/ml, and a portion of this solution is added to the mutagenesis reaction mixture. This diluted oligo stock should be discarded after use.

The first time you use this kit, you should do a practice with the control DNA template and oligos provided in the kit. This will allow you to see if the kit components and your technique are working. Typically we get > 100 blue colonies in the control reaction and > ~50 colonies in the mutagenesis reaction from your plasmid. If you get much less than 50 colonies you should try mutagenesis again using different temperature cycling conditions. In this case, increase the denaturing temperature from the recommended 95° C to 98° C, and change the annealing temperature from the recommended 55° C to 57° C, and increase the number of cycles from the recommeded to 20. We also transform the mutant DNA into the E. coli that comes with the kit. Then we prepare DNA from this strain for sequencing. We prepare DNA from 3 different colonies, and sequence 2 of them. After confirming the sequence, we take some of the purified plasmid from the plasmid prep from 1 colony and transform the E. coli strain desired for protein expression. A portion of the plasmid prep is also stored at -20° C.

You have the option to check the amplified DNA product prior to transformation. Simply load 5ml (not 10 ml as recommended by Stratagene) onto a 1% Agarose gel, and if you can see the amplified band with ethidium staining (it should have the length of the plasmid + insert) it is usually enough for transformation.

If you get much less than 50 colonies and you know the amplification worked (by Agarose gel analysis), you can try the transformation again or perhaps get a new batch of competent cells if they are old.

If you get no gel band, you can try changing the annealing temperature from 55° C to 57° C as noted above.

Sculptor Mutagenesis.

Preparation of Single-stranded DNA Template. We have found it best to make single-stranded DNA from plasmids propogated in JM109 E. coli. Your plasmid can be introduced into this strain by standard transformation. To make single stranded DNA from your plasmid, it must contain and f1 origin of replication (Be sure to check with the company that supplied the plasmid). The competent JM109 cells must be made by a special procedure using glucose/minimal medium. Grow JM109 on a glucose/minimal medium plate. These are made as follows: Make 1 liter of autoclaved 1X M9 salts with agar (6 g anhydrous Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 15 g agar). To this 1 liter, add 1 ml of autoclaved 1 M MgSO4, then add 1 ml of 1 M autoclaved thiamine-HCl, add 1 ml of autoclaved 0.1 M CaCl2, and add 10 ml of autoclaved 20% glucose. Pour the plates as usual. Store plates in cold room in a closed plastic bag or container. Pick a colony and grow it up and make competent cells in the standard way (see Maniatis manual for example). Transform your plasmid into competent JM109 as usual (see Maniatis manual for example) and then plate out on an LB plate containing the desired antibiotic.

In the early morning, innoculate 10 ml of TYP medium (1 liter contains 16 g Tryptone, 16 g yeast extract, 5 g NaCl, 2.5 g K2HPO4, autoclave) containing desired antibiotic with a colony of plasmid-transformed JM109 from the LB/antibiotic plate. Grow the culture at 37° C with shaking for 6 h, and add 0.5 ml of this culture to 5 ml of fresh TYP medium containing desired antibiotic, and culture for 1 h. Add 80 ml of helper phage R408 (Promega cat. # 229A). The phage comes as a suspension of 1011 pfu/ml, and we add the appropriate amount without titrating it in our lab. The phage stock can be stored at -20° C. Incubate the culture at 37° C overnight (12-13 h) with shaking. Transfer 1 ml of the 5 ml culture to each of 5 Eppendorf tubes.

To each tube, add 2 ml of DNAse-free RNAse (10 mg/ml stock, store RNAse stock at -20° C). Incubate 37° C for 15 min. Transfer the tubes to a 70° C water bath and incubate 15 min. Microfuge the Eppendorf tubes at ~10,000 g at 4° C (cold room) for 5 min. Transfer most of the supernatants to fresh Eppendorf tubes, be careful not to take the pellet (you can leave ~50ml of liquid in each tube). To each tube, add 200 ml of PEG/NaCl (20 g PEG-6000 (Sigma), 14.6 g NaCl in 100 ml water, then autoclave). Mix by briefly vortexing each tube, incubate 15 min at room temperature. Microfuge at ~10,000 x g for 5 min at 4° C (cold room). Remove all of the supernatant using a Pipetman, be careful not to place the tip at the bottom of the tube, sometimes you can see a faint pellet. Centrifuge again for 2 min and remove any remaining supernatant. Add to each tube, 100 ml of TE buffer, vortex 30 sec to resuspend the pellet. Add to each tube, 50 ml of TE-saturated phenol (phenol from Sigma) is swirled with TE buffer for a few minutes, let the layers settle and take the lower phenol layer, store this TE-saturated phenol at 4° C, the solution should not be yellow, if yellow make again with new bottle of phenol). Vortex each tube 20 sec, and incubate at room temperature for 15 min. Vortex for 20 sec, and microfuge at ~10,000 x g for 3 min at 4° C (cold room). With a Pipetman, transfer most of the upper layers to fresh Eppendorf tubes, avoid taking the lower layer or the interface which contains a film of protein. To each tube add, 100 ml of CHCl3/iso-amylalcohol (24:1, v:v), vortex 20 sec, microfuge at 10,000 x g for 4-5 min at room temperature. Use a Pipetman to transfer most of the upper layers to fresh Eppendorf tubes (don't take the lower phase or the interface). To each tube, add 10 ml of 3 M NaAcetate, pH 6.0, and add 250 ml of absolute ethanol. Incubate tubes in the -20° C freezer for 10 min, and microfuge for 15 min at ~10,000 x g at 4° C (cold room). Remove most of the supernatants with a Pipetman, being careful not to disturb the pellet (often invisible). To each tube of pellet, add 500 ml of freezer-cold 70% ethanol in water (store in -20° C freezer), vortex for few seconds, microfuge ~10,000 x g for 5 min at 4° C (cold room). Remove supernatant as above, and let the tubes sit open at room temperature for ~3-4 min to evaporate the liquid. To each tube, add 10 ml of TE, vortex for 15-20 sec and finally combine DNA from all 5 tubes into a single Eppendorf. Store single-stranded DNA at -20° C.

To quantify the single-stranded DNA, run a flat-bead gel of 1% Agarose in 1X TBE in the usual manner. Load 1-2 ml of single-stranded DNA stock in one lane. For a standard, load in a second lane 200 ng of double-stranded DNA from a plasmid-prep stock that has been quantified by fluorescence-dye binding assay. Stain the gel with ethidium bromide in the usual way and estimate the amount of single-stranded DNA by eye. Typically we get 4-5 mg of single stranded DNA. Since 2 mg is used to make each mutant, you may want to do a few single stranded DNA preps side-by-side so that you get enough DNA to make several mutants. This is important so you don’t have to keep remaking single stranded DNA.

Mutagenesis using single stranded template. Mutagenesis is carried out exactly as described in the Sculptor manual from Amersham using 2 mg of single stranded DNA. Important, the oligo used for mutagenesis must not contain an Nci I site because digestion with this enzyme is a required step in the procedure. You can always avoid adding this site by making a silent mutation. After the mutagenesis procedure, we transform XL1-Blue and isolate DNA from this transformant for DNA sequencing. Then transfer the plasmid to the desired bacterial strain for expression.

Making several mutations at one. One way that works well to make several mutations at once when the sites of mutations are close to each other is to use the Quickchange kit (Quick Change IIXL Site directed mutagenesis kit Cat#200521) to delete the region of interest and then to do a second round where you insert the region of interest with the mutations in place. For exmaple in the case of cPLA2, we have made a triple site mutation (at positions 541/543/544) by deletion of a small region of the gene using the primers:

fwd-del:

5'-TAT GAG CCT CTG GAT GTC AAA TTC ATG TAG TGG ACA GTG G-3'

rev-del:

5'-CCA CTG TCC ACT ACA TGA ATT TGA CAT CCA GAG GCT CAT A-3'

The clone was sequenced to be sure the deletion was made and then we inserted the mutated cassette using the following primers:

fwd-ins:

5'-TAT GAG CCT CTG GAT GTC AAC AGT AAC AAC ATT CAT GTA GTG GAC AGT GG-3'

rev-ins:

5'-CCA CTG TCC ACT ACA TGA ATG TTG TTA CTG TTG ACA TCC AGA GGC TCA TA-3'

and finally verified the insertion by DNA sequencing.

QuikChange has worked in most cases and should be tried first as it is the easiest and fastest to execute. One exception is for big plasmids like pVL for Baculovirus or some mammalian expression vectors. If the plasmid + insert is much above ~6000 nt you may want to put your insert into a small vector such as a pUC vector, make the mutant using QuikChange and then transfer the insert back to your desired plasmid. Of you can try the new modified QuickChange kit from Stratagene that is optimized for large plasmids. If QuikChange does not work, you can try the Sculptor kit. QuikChange starts with a double stranded plasmid, whereas Sculptor requires the preparation of a single stranded plasmid template, thus the QuikChange approach is faster. It is the policy of the Gelb lab, that the entire coding region of all mutated genes must be fully sequenced to verify not only that the mutation was introduced, but that there are no other mutations in the coding region. Finally we no longer buy the QuikChange Kit from Stratagene since it is much cheaper to buy the components separately and this works just fine.

The Pfu Turbo DNA polymerase is purchased from Stratagene. DpnI is from New England Biolabs or from the UW Biochemistry stockroom. The 10 mM dNTP stock is made from ATP,GTP,CTP, and TTP purchased at the UW Biochemistry Stock Room, the final concentration of nucleotide triphosphophates is 0.2 mM in the PCR reaction. If you want to do a positive control to be sure the mutagenesis is working, we have a stock stolution of the same beta-galactosidase plasmid that comes with the QuickChange kit (pWhitescript plasmid) and we also have the set of oligos needed to mutate this beta-galactosidase. For mutagenesis, follow the QuickChange manual and use the same concentration of components that Stratagene recommends. You should use XL1-Blue with electroporation for the transformation since not all E.coli strains have the capacity to repair the nicked plasmids generated from the QuickChange procedure.

Stragene Quik Change Manual

Design of oligos for mutagenesis. Two oligos are needed for QuickChange, whereas one is needed for Sculptor. We use the oligos as supplied by the company without further purification (despite the fact that Stratagene suggests gel purification of oligos for QuickChange, we have found this not to be necessary). For Sculptor but not for QuickChange, the oligo has to be 5'-phosphorylated.

For any kind of mutagenesis, you should check your oligos for stemloop formation and duplex formation with itself. To do this use the OLIGO program that runs on the PC in the lab. You simply type in the sequence of your oligo and it shows you any stemloop formation and any self-duplex formation. Avoid oligos that have extensive stemloop and/or self-duplex formation.

Oligos for OuickChange. The oligos should be ~30 nt in length (longer for multi-site mutations, see below). The 3' nucleotide should be a G or a C. The mutation region should be in the center of the oligo. The minimum GC content of the oligo is 40% (we have successfully used 40-75%). To make a single point mutation, try, as a first choice, to change only one nucleotide. If the genetic code does not permit this, we have been able to change 2-3 nucleotides in the codon with a single oligo. We have been able to make up to 3 amino acid changes with a single oligo. For this, the oligo should have 9-10 perfect match nucleotides at each end of the oligo. Here are two examples.

To make the R7E/K1OE/K16E mutant of human group IIa PLA2, we used the oligo:

GAA TTT CCA CGA GAT GAT CGA GTT GAC GAC AGG AGA GGA AGC CGC AC

The mutation sites are underscored. This is a 47-mer, with 10 nucleotides of perfect match before the first mutation site and 12 after the last mutation site.

For QuickChange, you also need to make the oligo that is the exact complement of the above oligos (i.e. not the wild type sequence but bearing the desired mutations).

Oligos for Sculptor. Use the same rules as for QuickChange oligos except for single-amino acid mutations, the oligo should be ~20 bp instead of 30 bp.

An example is the oligo to make the K124E/R127D mutant of human group IIa PLA2, we used the oligo:

CGG GGT CGA CCC ATC GCA GTG TTC GTT AGA GTA G

This oligo is a 34-mer with 13 nucleotides of perfect match before the first mutation site and 10 after the last mutation.

QuickChange Mutagenesis. We follow the exact procedure from Stratagene that comes with the kit except for the following change. We do the mutagenesis reaction at half the scale that the company gives (this saves reagents). Stratagene recommends using 5-50 ng of template DNA, but we always use 25 ng template DNA for half-size reaction (quantified using fluorescence dye binding assay, see Gelb lab handout). We use 62.5 ng of each oligo for half-size reaction. The oligos typically come in lyophilized form. We hydrate them with 200 ml TE buffer and store them at -20° C. An aliquot of this stock is used to quantify the amount of DNA by the OD260 (OD260 = 1, in a 1 cm path-length cuvete in a volume of 1 ml is 33 mg of DNA). A portion of the oligo stock solution is further diluted with water to give 50 ng/ml, and a portion of this solution is added to the mutagenesis reaction mixture. This diluted oligo stock should be discarded after use.

The first time you use this kit, you should do a practice with the control DNA template and oligos provided in the kit. This will allow you to see if the kit components and your technique are working. Typically we get > 100 blue colonies in the control reaction and > ~50 colonies in the mutagenesis reaction from your plasmid. If you get much less than 50 colonies you should try mutagenesis again using different temperature cycling conditions. In this case, increase the denaturing temperature from the recommended 95° C to 98° C, and change the annealing temperature from the recommended 55° C to 57° C, and increase the number of cycles from the recommeded to 20. We also transform the mutant DNA into the E. coli that comes with the kit. Then we prepare DNA from this strain for sequencing. We prepare DNA from 3 different colonies, and sequence 2 of them. After confirming the sequence, we take some of the purified plasmid from the plasmid prep from 1 colony and transform the E. coli strain desired for protein expression. A portion of the plasmid prep is also stored at -20° C.

You have the option to check the amplified DNA product prior to transformation. Simply load 5ml (not 10 ml as recommended by Stratagene) onto a 1% Agarose gel, and if you can see the amplified band with ethidium staining (it should have the length of the plasmid + insert) it is usually enough for transformation.

If you get much less than 50 colonies and you know the amplification worked (by Agarose gel analysis), you can try the transformation again or perhaps get a new batch of competent cells if they are old.

If you get no gel band, you can try changing the annealing temperature from 55° C to 57° C as noted above.

Sculptor Mutagenesis.

Preparation of Single-stranded DNA Template. We have found it best to make single-stranded DNA from plasmids propogated in JM109 E. coli. Your plasmid can be introduced into this strain by standard transformation. To make single stranded DNA from your plasmid, it must contain and f1 origin of replication (Be sure to check with the company that supplied the plasmid). The competent JM109 cells must be made by a special procedure using glucose/minimal medium. Grow JM109 on a glucose/minimal medium plate. These are made as follows: Make 1 liter of autoclaved 1X M9 salts with agar (6 g anhydrous Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 15 g agar). To this 1 liter, add 1 ml of autoclaved 1 M MgSO4, then add 1 ml of 1 M autoclaved thiamine-HCl, add 1 ml of autoclaved 0.1 M CaCl2, and add 10 ml of autoclaved 20% glucose. Pour the plates as usual. Store plates in cold room in a closed plastic bag or container. Pick a colony and grow it up and make competent cells in the standard way (see Maniatis manual for example). Transform your plasmid into competent JM109 as usual (see Maniatis manual for example) and then plate out on an LB plate containing the desired antibiotic.

In the early morning, innoculate 10 ml of TYP medium (1 liter contains 16 g Tryptone, 16 g yeast extract, 5 g NaCl, 2.5 g K2HPO4, autoclave) containing desired antibiotic with a colony of plasmid-transformed JM109 from the LB/antibiotic plate. Grow the culture at 37° C with shaking for 6 h, and add 0.5 ml of this culture to 5 ml of fresh TYP medium containing desired antibiotic, and culture for 1 h. Add 80 ml of helper phage R408 (Promega cat. # 229A). The phage comes as a suspension of 1011 pfu/ml, and we add the appropriate amount without titrating it in our lab. The phage stock can be stored at -20° C. Incubate the culture at 37° C overnight (12-13 h) with shaking. Transfer 1 ml of the 5 ml culture to each of 5 Eppendorf tubes.

To each tube, add 2 ml of DNAse-free RNAse (10 mg/ml stock, store RNAse stock at -20° C). Incubate 37° C for 15 min. Transfer the tubes to a 70° C water bath and incubate 15 min. Microfuge the Eppendorf tubes at ~10,000 g at 4° C (cold room) for 5 min. Transfer most of the supernatants to fresh Eppendorf tubes, be careful not to take the pellet (you can leave ~50ml of liquid in each tube). To each tube, add 200 ml of PEG/NaCl (20 g PEG-6000 (Sigma), 14.6 g NaCl in 100 ml water, then autoclave). Mix by briefly vortexing each tube, incubate 15 min at room temperature. Microfuge at ~10,000 x g for 5 min at 4° C (cold room). Remove all of the supernatant using a Pipetman, be careful not to place the tip at the bottom of the tube, sometimes you can see a faint pellet. Centrifuge again for 2 min and remove any remaining supernatant. Add to each tube, 100 ml of TE buffer, vortex 30 sec to resuspend the pellet. Add to each tube, 50 ml of TE-saturated phenol (phenol from Sigma) is swirled with TE buffer for a few minutes, let the layers settle and take the lower phenol layer, store this TE-saturated phenol at 4° C, the solution should not be yellow, if yellow make again with new bottle of phenol). Vortex each tube 20 sec, and incubate at room temperature for 15 min. Vortex for 20 sec, and microfuge at ~10,000 x g for 3 min at 4° C (cold room). With a Pipetman, transfer most of the upper layers to fresh Eppendorf tubes, avoid taking the lower layer or the interface which contains a film of protein. To each tube add, 100 ml of CHCl3/iso-amylalcohol (24:1, v:v), vortex 20 sec, microfuge at 10,000 x g for 4-5 min at room temperature. Use a Pipetman to transfer most of the upper layers to fresh Eppendorf tubes (don't take the lower phase or the interface). To each tube, add 10 ml of 3 M NaAcetate, pH 6.0, and add 250 ml of absolute ethanol. Incubate tubes in the -20° C freezer for 10 min, and microfuge for 15 min at ~10,000 x g at 4° C (cold room). Remove most of the supernatants with a Pipetman, being careful not to disturb the pellet (often invisible). To each tube of pellet, add 500 ml of freezer-cold 70% ethanol in water (store in -20° C freezer), vortex for few seconds, microfuge ~10,000 x g for 5 min at 4° C (cold room). Remove supernatant as above, and let the tubes sit open at room temperature for ~3-4 min to evaporate the liquid. To each tube, add 10 ml of TE, vortex for 15-20 sec and finally combine DNA from all 5 tubes into a single Eppendorf. Store single-stranded DNA at -20° C.

To quantify the single-stranded DNA, run a flat-bead gel of 1% Agarose in 1X TBE in the usual manner. Load 1-2 ml of single-stranded DNA stock in one lane. For a standard, load in a second lane 200 ng of double-stranded DNA from a plasmid-prep stock that has been quantified by fluorescence-dye binding assay. Stain the gel with ethidium bromide in the usual way and estimate the amount of single-stranded DNA by eye. Typically we get 4-5 mg of single stranded DNA. Since 2 mg is used to make each mutant, you may want to do a few single stranded DNA preps side-by-side so that you get enough DNA to make several mutants. This is important so you don’t have to keep remaking single stranded DNA.

Mutagenesis using single stranded template. Mutagenesis is carried out exactly as described in the Sculptor manual from Amersham using 2 mg of single stranded DNA. Important, the oligo used for mutagenesis must not contain an Nci I site because digestion with this enzyme is a required step in the procedure. You can always avoid adding this site by making a silent mutation. After the mutagenesis procedure, we transform XL1-Blue and isolate DNA from this transformant for DNA sequencing. Then transfer the plasmid to the desired bacterial strain for expression.

Making several mutations at one. One way that works well to make several mutations at once when the sites of mutations are close to each other is to use the Quickchange kit (Quick Change IIXL Site directed mutagenesis kit Cat#200521) to delete the region of interest and then to do a second round where you insert the region of interest with the mutations in place. For exmaple in the case of cPLA2, we have made a triple site mutation (at positions 541/543/544) by deletion of a small region of the gene using the primers:

fwd-del:

5'-TAT GAG CCT CTG GAT GTC AAA TTC ATG TAG TGG ACA GTG G-3'

rev-del:

5'-CCA CTG TCC ACT ACA TGA ATT TGA CAT CCA GAG GCT CAT A-3'

The clone was sequenced to be sure the deletion was made and then we inserted the mutated cassette using the following primers:

fwd-ins:

5'-TAT GAG CCT CTG GAT GTC AAC AGT AAC AAC ATT CAT GTA GTG GAC AGT GG-3'

rev-ins:

5'-CCA CTG TCC ACT ACA TGA ATG TTG TTA CTG TTG ACA TCC AGA GGC TCA TA-3'

and finally verified the insertion by DNA sequencing.