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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

 

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!

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.

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.

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