Are you using any Kits to do the Site-Directed mutagenesis? What did you mean double primers? Is it multiplex PCR? I think the primers are giving amplicon which is linear product. Try to check the primers again.

Hi Anchal...first of all...the tm of your primers is too low..if you read in SDM kit manual..they strictly say that the tm should be >75. the extension for your pfu turbo is at 68, which is close to 60, so it will not be very efficient. But since you were able to see amplicons, there can be a problem with your competent cells. How much you are transforming?? generally out of 50ul, 10ul is used for checking the amplicon and rest is used dpn. so out of 40ul, transform 2ul only. Try to prepare fresh competent cells or make electrocompetent cells, which are more efficient.

Your may be having problem with transformation....

Are you doing what is essentially a Quick-Change type SDM protocol? We have much better luck (and can simplify our mutagenic primer pool) by doing a modified, megaprimer-based quick-change protocol (MEGAWHOP)l. The megaprimer step allows you to verify that your primers amplify correctly, and you only need one mutagenic primer along with one of your existing gene-flanking primers. For a complete description of our protocol, see http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Recombinant+DNA+Protocols#Site_directed_mutagenesis_by_Megaprimer_Quick_Change_MEGAWHOP_PCR.

Cheers.

Yes I agree with Andaleeb, it may be a efficiency problem in the cloning. In SDM there are two PCR products to be cloned at once so ratios on insert and vector should be optimal (3-5/1). I used to electroporate the ligation reaction in this kind of cloning.

Which SDM system and which host E.coli cells did you use for transformation? As per my knowledge, not all E.coli strains work with the PCR product transformation. They should have a mechanism to ligate and circularise the linear PCR product before amplifying it. If your host cell does not have this mechanism then you would not get positive clones on antibiotic plate. If cells are with the kit itself then they should work and in that case there could some other problem, may be frameshift mutation or something. did you sequence your construct?

For transformation of PCR product, its concentration is also crucial so you would look into these parameters as well.

Hope this is useful.

Good luck with the experiment!

Your problem is likely in transformation step because you see amplicons after DpnI digestion (DpnI will digest all template plasmids... so the remaining plasmids you see as amplicons are the one PCR amplified...if you had given adequete digestion time+enzyme) So its very likely your trouble is in transformation. For transformation of SDM plasmids you have to use NZY+ broth. See the technical manual in the following link and follow exactly the transformation protocol using blue white selection+NZY+ broth. You should get the mutants. The manual link is http://www.tufts.edu/~mcourt01/Documents/Stratagene%20Quikchange%20mutagenesis.pdf

Good Luck..!!

I was thinking that if you digested the PCR products with DpnI check how many extra nucleotides at the ends of the amplicon you need to have so de enzyme is able to cut. There are tables about this in the New England Biolabs.

Most likely is a problem with the competency of the cells in the transformation. I suggest also to use a modified formula to calculate the melting temp that is in the QuikChange Site-Directed Mutagenesis Kit [Tm = 81.5 + 0.41 (%GC) - 675/N - % mismatch], it usually works for me.

Hello Anchal,

Since this is most likely a problem with the transformation step, I wonder if you inactivated DpnI after digestion and if you dialysed your sample prior to transformation.

Also, if the problem is due to low competency of your E. coli you may add calcium chloride to 0.1M to improve competency.

Good luck.

I find that doing a ligation on the PCR product with regular DNA ligase (I use promega T4 DNA ligase) before transformation sometimes helps.

@ Bastian: I had digested 20ul of pcr with 1 ul of dpn. for 6-8 hrs at 37 degrees..incubator... my water bath...had some issues...What was the efficiency of ur comp cells

@ all : thanks for the replies... my mutagenesis worked on 15/2/2013...the date is etched in my memory nw..the problem was competent cells...

Hey.

I do have the same problem when I do SDM. After Dpn digestion, when I run in agarose gel, I could see only primers. I cannot see any of my PCR product. Is there anything wrong in my PCR amplication ? PS : I tried with all different Polymerases (pfu, pfu turbo, ultra) but I dint get my product.

Kindly advise. Thanks

Hello Ya Yesh,

I would make sure that the primer design is correct. Try our software tool for primer design: http://primergenesis.com

You can also fins some helpful tutorials here: http://www.primergenesis.com/Validation.php

Hope this helps. Let us know if you have more questions :)

Bruno

Hey guys! in my case I did not design the primers via any software. I Had to mutate two aminoacids separated by two aminoacids from just one primer pair and it worked. I used Herculase. The primer length was around 60bp. One has play around with Mg conc: and additives such as DMSO and also the annealing temp:. In my case 2.5mM extra Mg and an initial annealing at 45C for 5 min and then 65C worked fine. Hope this might help some of you guys.

Hi Anchal,

I strongly suspect on your primers whether it is designed manually or by strategene online software? The SDM primers are invert primers which amplifies the whole plasmid, if there is a problem in primers still results in linear amplicon but not the single strand circular plasmid. Secondly, Dpn1 digestion should be for short time... I found that you are given for 6 -7 hours that can have star activity by chopping the likely-site in your plasmid and linearizing it. one hour digestion is more then sufficient for disrupting the methylated parent strand.

Our free site-directed mutagenesis software tool moved to http://primergenesis.org

Happy mutagenesis!

Bruno