Hi Asma,

I assume you are using QuikChange from Agilent? If so, it seems a bit strange that you use dNTP and polymerase from other companies. I know that, in theory, everything should work if you substitute components in the kit with the right material, but in practice, it's not always the case. I was trying to follow the QuikChange protocol using polymerase from another company, and I just couldn't get it to work. As soon as I use everything from the kit, BAM, I got bands on my agarose gel. My first advice is to use everything from the kit, and my second advice would be to add DMSO, which is labelled QuikSolution in the kit. The manual suggests titrating up to 1.5 ul of QuikSolution in your reaction. Hope it helps.

Best regards,

Arthur

P.S. FYI, the kit that I used was QuikChange Lightning.

I have two suggestions. 

1) Please check if your polymerase is suitable for SDM. Some of the polymerases with high proofreading activity are unsuitable for SDM (like Phusion, for example). 

2) Consider adding DMSO at 5% in your PCR reaction. 

And, as suggested by Adam, longer primers for SDM are always better. 

Best wishes, 

Santosh. 

The problem lies in the primers, where the primer sequences are complement each other as said by Zeeshan. I have worked with same problem, first i designed complementary primers, did n,t workout for me for a month or more time. Later I designed overlap primers, where your primers overlap only for 12 bases. total primer length should be 33-36 bases. I see the number of cycles can be reduced from 25 to 18 and use high fidelity buffer supplied along with mgcl2 (Final concentration 1X).

Change primers, it will work immediately, from personal experience.

I had success with split PCR : PCR#1 with forward primer only, clean up the product and use it in PCR#2 with reverse primer.

Use Quick Change XL reaction system, which is suitable for bigger plasmid/DNA template.  .

Dear All,

Thanks for your comments.

I added DMSO (3% and 5%) in my PCR reaction but nothing changed! This primer is designed for a single mutation and changed codon is in the middle of the primer nearly. I tried spilt PCR with forward and reverse primer separately for 10 cycles and then mixed them for 15 cycles but not any result.

I'm really confused! When I use another mutagenic primer that was designed for the other site of gene, I can see PCR product on the gel! In theory, my primer has optimum physicochemical properties. I do not know what to change it.

Your primers are too well matched.  There are a couple of options.  You could design two new complimentary primers about half way on other "side" of the vector and do two PCRs using one old primer with a new primer to generate two halves of your plasmid and then use the PCR products in a gibson assembly.  You could use the quikchange multi mutagenesis kit and only use one of your primers for the reaction.  You could redesign your primers adding ~10 bases to 3' of your forward and reverse primers.  Long PCR can be tricky you might need some additives and optimization.

What type of mutation are you trying to put in?  I think the primers are fine if you are changing 1 or 2 nucleotides.  I have used complementary primers many times before for simple point mutations.  Once I tried to introduce an entire amino acid deletion using complementary primers and it did not work.  I then tried staggered primers and it was successful.  (staggered primers paper: Liu and Naismith 2008 BMC Biotechnology)  

You should also try using less template.  You are using 140 ng and the QuikChange protocol calls for 10 ng.  Have you tried varying the amount of your template (5-50 ng) as suggested in the manual? 

Also, and probably more importantly, you should make sure your polymerase is compatible or simply use the PfuUltra polymerase that is optimized for  the QuikChange kit (as some of the others have already suggested). 

Your elongation step is 11 minutes long so I assume your plasmid is 11 kb.  This seems pretty long so you really should be using the QuikChange II XL kit.  I have typically worked with plasmids in the ~8kb range and I have used the XL kit with success. 

If I were you, I would start over using the XL kit and follow the instructions exactly. 

Also, did you design the primers using the Agilent software? 

http://www.genomics.agilent.com/primerDesignProgram.jsp

You have to register and long in but I have found this works extremely well.  When I used another online primer designing program, only about half of the primers worked.  I have never had a primer not work after using the Agilent program. 

Try 500C-550C Gradient PCR for annealing.. Most of my SDMs worked at around 52 degrees Centigrade.. All da best!!

I'm experiencing the same problem here trying to introduce HA tag in the N and C-termini of ToMMV iCDNA or about 6.5kb. Not a single band but the primers are ok. I am confused. I will try some of the recommendations here.

I am having similar issues with my site directed mutagenesis. These are my primers (capitalized is the mutation I am introducing)

F- catcccagtcaagcagaacGCGttcaccgtcaaccgct

R-agcggttgacggtgaaCGCgttctgcttgactgggatg

I am using 25uL reaction mix

template-70ng

5X buffer-5uL

10X dNTP-2.5uL

DMSO-5uL

0.5uM R and F

0.5uL Taq polymerase

PCR conditions

98deg -30s (initial denaturation)

98deg-30s

55deg-1min (annealing)

72deg-13min (extension )

steps 2-4 repeated 14 cycles.

final ext 5min

I am trying to amplify 13kb plasmid. I keep getting primer dimers and no pcr product.

I am facing a similar problem. While using the overlap PCR method, whenever I'm trying to anneal two fragments (A-1700bp, B-900bp), I'm getting a final product band at 900bp (i.e. only band B is getting amplified). The total size of my product should have been 2600bp.

Can anyone please help me to troubleshoot this problem?

Note: I'm not using any kit or phosphorylated primers/T4 ligase.