Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Site directed mutagenesis stopped working. - (Oct/25/2016 )

Hi folks,


It has been 4 months now since my PCR for site directed mutagenesis (SDM) stopped working. I started in June 2016 , transformed after PCR and obtained colonies that contained my mutant. The first month was great. I don't have any idea what kinda hell launched itself on my PCR, it just stopped working.  I use the following protocol:-


4ul 5 HF buffer

20ng template

125ng forward and reverse primers

0.4ul dNTP 

0.2ul Polymerase

and making the reaction upto 20ul usingddH20


PCR programme:-


98C - 30 sec

Cycle 16X

98C for 15 sec

69C for 60sec

72C for 13 min


Final extension

72C for 15 min

Hold 10C indefinitely


product : 20Kb


The issue is that this protocol worked fine and then stopped working. I have tried adding DMSO but to no avail. Also, tried running more cycles, running the PCR products on gel, NOTHING !!!!!!!!!!!!!!!!!

I have also made new primer aliquots from the stocks, NOTHING!!!!!!!!!!!

It has reached a frustration point now ,a s i have'nt got any data since June.


PLEASE HELP!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!


-Akash Deep-

ALso, i should mention that transformation protocol works fine as i have been doing a lot of transformation with other products. Therefore, it is the PCR which is the bug!

-Akash Deep-

are you sure that all of your pcr components are the correct concentration (especially the dntps)? that the polymerase is still active?


Yes. The concentration of the components are correct. I have also tried using new dNTP's and polymerase.

What bugs me is that it was working fine but then it stopped working.

-Akash Deep-

have you tried using a different thermal cycler?


have you tried other pcr sets (template and primers) or a control reaction?


Yes , i have used a different thermal cycler.

My lab mate uses the same kit for some of her reactions and they seem to work fine. I have tried increasing cycle numbers , changing template concentrations,but no effect.

-Akash Deep-

Are the failed reactions specific to your template? In other words, can you make a different mutation on the same template using identical reagents?