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Help! Problems with SDM with QUikChange II XL site-directed mutagenesis kit


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#1 eyeboy

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Posted 14 April 2011 - 10:16 PM

Hi guys, just joined to see if I could get any input into this problem I have with site directed mutagenesis and subsequent competent cell transformation.

I'm using the Stratagene QUikChange II XL Site-Directed Mutagenesis Kit, with primers designed using the program on Stratagene's website.

The vector that I am doing site-directed mutagenesis on is a pApex-3P-hTERT-SV40-puro vector (~12kBp in size) and the hTERT sequence (where the mutagenesis is located) is very GC rich, hence the primer melting temperature is in the vicinity of 90 degrees celcius.

However the kit is designed to deal with such parameters by having the QuikChange solution containing DMSO which helps prevent secondary structures from forming and decreasing the efficiency of the mutagenesis reaction.

The things I've tried include varying the amounts of template, primers, QuikCHange solution, adding additional DMSO and the annealing temperatures. I've found that by having DMSO of more than 10% results in a smearing effect when viewing the products on agarose gel. With optimal product forming between 4-8% DMSO. And I know that I've gotten rid of parental strand DNA because I used the DpnI restriction digest and if there were incomplete digest, I would see the plasmid band in every sample. But I don't, only strong products in samples containing 4 and 8% DMSO.

HOWEVER, when I try to transform them into competent cells from the kit, I just dont get colonies.

Anyone with any similar experiences willing to help me out please??

Any help would be greatly appreciated

#2 jaya2020

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Posted 18 April 2011 - 02:55 AM

Hi,

Did u try the controls. Try the control and it should work fine. Hope the conditions in the PCR is according to the manufactures instruction.
The quick solution in the kit is DMSO. There is no need to add additional DMSO to the reaction.
Check the concentration of the primers. It should be exactly 125ng, not more not less.
Run the product on a gel. If u r reaction is working u should the see the band after Dpn I digestion, although may be faint. I generally use 10ul of the reaction product on the gel. If the reaction didnt work, u should be able to see the primer dimers which would be less than 0.4kb on the gel.

I would recommend to do the control first. Also see to that the primers are designed correctly. Good luck

jaya

#3 jaya2020

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Posted 18 April 2011 - 02:57 AM

Hi,

Did u try the controls. Try the control and it should work fine. Hope the conditions in the PCR is according to the manufactures instruction.
The quick solution in the kit is DMSO. There is no need to add additional DMSO to the reaction.
Check the concentration of the primers. It should be exactly 125ng, not more not less.
Run the product on a gel. If u r reaction is working u should the see the band after Dpn I digestion, although may be faint. I generally use 10ul of the reaction product on the gel. If the reaction didnt work, u should be able to see the primer dimers which would be less than 0.4kb on the gel.

I would recommend to do the control first. Also see to that the primers are designed correctly. Good luck

jaya

Also check the efficiency of the competent cells by having a positive control of ur sample. Hope it should work

jaya




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