QuickChnage Mutagenesis problem - no mutation, please help!!!!!!!! (Jan/02/2008 )

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Hi all, thank you for your advise, i did again the procedure, this time i did 40 cycles, and a digestion overnight with DpnI, and also I reduce the amount of template. At the end, when i transform it, i had like 12 colonies in total... I a waiting for the sequence to look for the mutations, but i want to show you guys how th gel of the PCR looks like, so may be you can tell me if it is good or not.
Thank you

QUOTE (vairus @ Jan 5 2008, 05:55 AM)

Try to do the PCR for 40 cycles to see if you get any amplification at all. I would strongly recommend using 55°C as starting annealing temp and to increase your annealing time from 10 seconds to 30 or even one minute.
What e. coli strain is your plasmid isolated from (100% sure it's methylated?).
If you have accuprime pfx, you might try it, maybe you're more succesfull with this enzyme (though pfu's work very fine). Also: dilute your template, I never go above 30 ng for mutagenesis reactions, mostly I use 5-10 ng. Increasing cycles from 18 to 25 might also help.

Good luck.

-MCR-

It looks like your PCR products are too small, but if you have colony's you might be lucky and have the right clone.

-vairus-

Hi, Thank you so much for all the advise!!!!!! i have my mutations finally!!!!! i recived the sequencing of my colonies, and looks ok, with the mutations... now i am going to transfect it in some 293T cells to do a functional analysis!!!!!!!!!

THANKS

QUOTE (vairus @ Jan 11 2008, 02:53 AM)

It looks like your PCR products are too small, but if you have colony's you might be lucky and have the right clone.

-MCR-

QUOTE (MCR @ Jan 15 2008, 02:55 PM)

Hi, Thank you so much for all the advise!!!!!!

i have my mutations finally!!!!! i recived the sequencing of my colonies, and looks ok, with the mutations... now i am going to transfect it in some 293T cells to do a functional analysis!!!!!!!!!

THANKS

QUOTE (vairus @ Jan 11 2008, 02:53 AM)

It looks like your PCR products are too small, but if you have colony's you might be lucky and have the right clone.

Hi,
Would you like to tell me how many bp of your primers?
Thanks

-andey-

This is the original ligation-during-amplification (LDA) method paper by Chen and Ruffner that these site-direct mutagenesis kits are based or derived from. It should help solve your problem.

-chessplayer-

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