Site-directed mutagenesis failed - (Dec/10/2013 )

hello everybody,

 

let me share my frustration :)

 

i would like to make a site-directed mutagenesis on a vector but it has been failed again and again.

 

I mean I have never got the amplified mutant plasmid.

 

here is my protocol:

 

- x ul plasmid template (50 ng)

- 1 ul FW primer (10 pmol/ul)

- 1 ul REV primer (10 pmol/ul)

fully complementer to each other (36 bp , Tm: 83 C)

- 5 ul buffer (10X)

- 1 ul Pfu ultra II polymerase

- 1,5 ul dNTP mix (10 mM)

- x ul H2O

 

Vt: 50 ul

- - -

denaturing temperature: 95 C

annealing temperature: 55 C

extension temperature: 72 C

25 cycles

 

I don't know the "hidden factor" that wasn't to be considered...

 

SF

You don't say what the result is. Do you get transformants that are original vector? Do you get no transfomants?

 

I'd say you should dramatically lower the template concentration. After PCR, you should digest the DNA product with DpnI to eliminate template DNA. Both of these will help you suppress transformed cells with the original plasmid sequence.

Hello phage,

 

I didn't perform the DpnI digestion then the transformation cause I didn't get PCR product. That's the basic problem.

i have a feeling my suggestion could be wrong but here it is.

check if its the right plasmid. primer/sequence.

some inhibitory factor in one of the extracts is inhibiting the reaction possibly.

i usually use 34 cycles

do you have any primer dimers?

what do you mean by complete complimentarity.?

use a different polymerase or and buffer.

hope u resolve it.

I assume the mutation you are attempting to insert is n the middle of the 36 bp. How confident are you that the plasmid contains the matching sequence (except for the mutation you need to insert)? How are you determining that your PCR fails to work? With 50 ng of template, you will always get a band at the correct length.

Thank you for your comments, guys ! It is sure that the plasmid contains the matching sequence - I have sequence results. I thought that PCR didn't work cause I only saw the template plasmid at the correct lenght. I assume if I have a successful PCR reaction the result is a very bright band not the template band of 50 ng DNA.

 

Do you think that primer concentration is fine (10 pmol/ul) ?

For SDM protocols the PCR step is NOT an amplification step, it is about replicating the plasmid DNA so that you can then digest away the methylated parent DNA. 

 

In other words - Do the DpnI digest....

hi this answer by bob 1 makes sense..

 

For SDM protocols the PCR step is NOT an amplification step, it is about replicating the plasmid DNA so that you can then digest away the methylated parent DNA. 

 

In other words - Do the DpnI digest....

Thanks guys ! It really makes sense. What a stuped I am :D

Hello everbody,

 

I would like to know opinions about my question: is it possible to make SDM with long primers (50-100 nt) that have high Tm ? Tm 90 or 100 C primers, for example. With a relatively high annealing temperature and the use of DMSO for avoiding secondary structure of the long primers.

 

SR