Hi Sumit,

Did you tried to transform the PCR-reaction? It is not uncommon that there is no PCR band, since the amount of DNA used in the mutagenesis is too low for being seen on agarose gel....We have done many side directed mutagenesis with this kit and i was quite successful even when no band was seen...just try it.

Hello Sumit!

Here are some ideas that might help you with your problem:

1.) What is the origin and purity of the template? (was it purified by using a plasmid DNA isolation kit?)

What is the host E. coli strain? Some strains are dam- (DNA methylation negative)

and cannot be used for synthesizing the template DNA needed for the mutagenesis reaction.

2.) The primer design software sometimes produces unsatifactory results that need

manual editing. I usually try to get two or three G or C at both ends (if possible).

3.) Double check that you used the correct selection marker in your experiment.

4.) I checked the manual for the QuikChange II kit and found different timings for the cycling reactions: 95 °C 30 sec; (18x) 95 °C 30 sec, 55 °C 1 min, 68 °C 8 min (for an 8 kbp plasmid), no final extension needed. Which version of the kit do you use?

Hope this helps.

Best regards,

Christian

Hi Sumit,

I often find that when i am amplifying templates as large as yours (8kb) it often helps to design primers manually, as Christian suggested, to contain 2-3 g/c bp at both the 3'and 5' ends of the oligonucleotides but also with quite high annealing temperatures (~60-70 °C but not as high as stratagene suggest 78°C). The reason for this is that given the size of the template there is a much higher probability of non specific primer annealing and formation of primer dimers, which can be detrimental to PCR efficiency (especially when using the stratagene quickchange kit that advises you to have a primer Tm of >78°C but to use an annealing temperature of 55°C.) By increasing the annealing temperature to within 5°C of yout primer Tm you may reduce the primer dimers that you see in your reaction and improve correct template annealing.

Or alternatively you could use a different SDM kit. I prefer to use the Phusion HF polymerase SDM kit from Thermo Scientific, although this does require that you perform a ligation step prior to transforming and that you start with high purity (RP-HPLC/PAGE) phosphorylated primers (these can be bought as standard from any oligonucleotide supplier)

I hope this helps a little!!!

Many thanks,

James

Hi James,

I have designed another pair of primers that are 5' back to back. Each primer has one mutation towards 4 bases downstream of their 5' ends (since I want to introduce two point mutations in my template). The primers are 24 bases long. I am gonna use Phusion polymerase kit from Thermo. Do you think 4 matching bases upstream of the bubble would be too short? Actually my mutated sites are 10 bases apart on the template. So I designed my primers this way.

Thanks

hi Christian,

I isolated my plasmid using Qiagen kit. Although my yield was quiet high (540ng/ul for a 50 ul elution), when I ran this sample in a gel, I found supersoild as well as some amount of linear and nicked DNA. Initially I was very surprised at the yield I got. I also did a single restriction digestion, and I could see 100% linear fragments. Also, for the cloning I used JM109 which I know is Dam+.

I am very sure that I am using the right selection as I always transform one set with my parental plasmid, and I see colony growth.

I tried the reaction set time times specified by strata gene, however that did not work. Unfortunately I am out of Pfu Ultra (by the way I was using Quick Change II kit). Now I have Pfu Turbo. So, I am using this along with the buffer that came with the Quick Change II kit (although meant for Pfu Ultra). Do you think this might be an issue?

Hi Sumit,

You may find it better to place one of your mutation sites in the middle of the first primer and the second mutation encoded at the very 3'end of the next, this would give you more flexibility to have more bp either side of the mutation site in your first primer (as 4 bp is a little short). See how your new primers go first then go from there.

The lenght of your primers (24 bp) is not so much of an important factor but more so it is the Tm of the primers that are important. As long as the primer pairs have a similar Tm and are relatively high (65-72 degC) to avoid non specific binding elsewhere in your plasmid you should be fine.

Good luck and please le tme know how you get on or if you have any other questions.

All the best,

James

Hi, maybe your mutations are way too apart for having good efficiency in primer annealing. I suggest you insert both mutations in a 2-step process.

Design 1 pair of 22-24bp primers with only one of the mutations in the middle of the sequence and also with 2-4 G/C at the ends and do the mutagenesis as described in the kit (54-55 annealing temp), then the DpnI, tansformation and miniprep.

Once you get your single-mutant primer, use it as template and repeat the process with a new set of primers for the second mutation.

I know it takes about 2-3 extra days but it is a safe bet since it should work with no problems.

Good luck.

> So, I am using this along with the buffer that came with the Quick Change II kit (although meant for Pfu Ultra). Do you think this might be an issue?

I think that this could be an issue. Your plasmid is quite large and I am not sure if Pfu Turbo is up to the task of synthesizing the complementary strands in the 'Quikchange reaction'. Maybe you can get some Pfu Ultra from another lab at your university?

I would recommend increasing to 28-32 cycles. At 18 cycles you may not have enough product to visualize.

Also, I prefer to do site-directed mutagenesis cloning in smaller vectors, then copy the mutated sequence into the expression vector.

I have always had good results with QuikChange Mutagenesis kits and primers designed using Agilent's primer design program. Even at 8bp apart, you can use overlapping primers. I have routinely had to use primers 40-50 bp long with no problems.

Hi Sumit,

I actually made my own kit. I bought QIAgen HiFi polymerase kit and Dpn1 enzyme. I order 2 complementary primers that are 31 bp each with mutation directly in the middle. I don’t care about the sequence. Important point: primers have to be HPLC or PAGE purified (it won’t work otherwise), plasmid freshly purified and with high 260/230 ratio. Than I do 2 separate 25 microL PCRs. One with forward primer and one with reverse for 10 cycles (5 min – activation, 15 s denaturation, 50C 30 s and 68C 2min/1kb). After 10 cycles I combine both reactions and add additional 0,75 microL HiFi and perform additional 18 or 20 cycles to assemble the whole thing. After that, direct Dpn1 digest 17 microL of PCR mix, 2 NEB buffer and 1 microL of Dpn1 for 2 to 3 hours. After that I transfect in E. coli using TransformAid (Thermo Fisher). I don’t check the PCR, I do control for plasmid degradation and I usually get 10 to 20 colonies – more than plenty to find your plasmid with mutation. I usually give that to my undergrad students to teach them PCR and mutagenesis and it always worked after second or third try.

Hi Sumit,

Did you tried to transform the PCR-reaction? It is not uncommon that there is no PCR band, since the amount of DNA used in the mutagenesis is too low for being seen on agarose gel....We have done many side directed mutagenesis with this kit and i was quite successful even when no band was seen...just try it.

Sumit,

I ran into a similar issue when trying site directed mutagenesis on a 8kb template. Does your template have high GC content or many mononucleotide repeats? If so, I would try Q5 polymerase from NEB. Although I could not get the mutagenesis to work, that polymerase came close to working. I was getting products of incorrect size though instead of blank gel. In the end I went with the tedious process of cloning small fragments (1-2kb) and piecing them together using restriction enzyme sites placed at the ends.

Also try lower annealing temperature. I sometimes go as low as 48C for tricky mutations. and usually 30 cycles

Hi Sumit,

we have often encountered problems with this approach when trying to introduce two or more mutations simultaneously. As others have advised, you may actually save time by making the mutations in sequential steps. Alternatively, if suitable restriction sites flank the region you want to mutate, why not just ligate in an ultramer containing your two point mutations.

Hello Everyone,

I really appreciate all of your valuable suggestions! I really learned a lot about the different ways that I can troubleshoot the issue that I was having.

Fortunately, this time I didn't have to dig a lot to get my experiment work. I just increased my template concentration to 150ng and did the site directed mutagenesis with the Agilent kit. I ran the thermo cycler for 16 cycles and then without checking the amplified product in an agarose gel carried out the transformation...:). Got colonies, sent a couple for sequencing and got the mutated ones!!!!!!!!!! It worked!!!!!!!

Thanks again to all of you for sharing your thoughts!

If you have still have any problem. please see my paper where we have incorporated many mutation in single PCR

Article Single-step overlap-primer-walk polymerase chain reaction fo...

after mutagenesis, there is no need to run your sample on agarose gel (conc. too low). it is good to directlty transforn 5-8 microlitres of reaction mixture.....

I have the same size of the plasmid as your, but i had used slightly different PCR condition 95 for 30sec, 24 cycle of 95 for 30sec, 60 for 1min and 68 for 8 to 10 min and 68 for 5min. The primers were used are of 25bp long becoz initially my PCR doesnt worked with long primer (35bp) highly repeated sequences.