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site directed mutagenesis


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#1 ayse

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Posted 24 January 2005 - 11:29 AM

Hi,
i'm trying to get point mutation using stratagene's quickchange site directed mutagenesis kit . my insert is 1.85 kb and is cloned into the polylinker of the eukaryotic expression vector pRcCMV. The control is working very fine and normally I didn't have any problems obtaining the blue colonies. However, the Tm of some of the primers I should use are nearly 80 degrees and their GC contents are %75-90. With these primers the numbers of the blue colonies that i obtained were very low and i also detected a large amount of primer-dimers. I have increased the annealing temperature, and tried 55, 60 and 65 degrees but I still have no decrease in my primer dimers. I also increased the DNA amount that I'm adding but it didn't help me too. Does anyone has any other suggestion?
Thanks

#2 ros

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Posted 01 February 2005 - 02:17 PM

i dont think your primer dimers should be affecting your colony number because they shouldnt contain the antibody selection gene that the whole plasmid contains, and hence should not be able to grow on antibiotic plates.

when you were designing the primers, did you follow the guidelines of the stratagene kit? it gives pretty detailed instructions. the Tm is supposed to be around 80 degrees anyway.

and did you get your primers PAGE of HPLC purified? it's supposed to increase the efficiency of the mutagenesis. dont know if this is true, but so far PAGE purification (Sigma-Genosys probes) has worked for me (although it is more expensive).

#3 Beautiful_Tits

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Posted 01 February 2005 - 02:33 PM

i dont think your primer dimers should be affecting your colony number because they shouldnt contain the antibody selection gene that the whole plasmid contains, and hence should not be able to grow on antibiotic plates.

when you were designing the primers, did you follow the guidelines of the stratagene kit? it gives pretty detailed instructions. the Tm is supposed to be around 80 degrees anyway.

and did you get your primers PAGE of HPLC purified? it's supposed to increase the efficiency of the mutagenesis. dont know if this is true, but so far PAGE purification (Sigma-Genosys probes) has worked for me (although it is more expensive).

Yep, I agree

#4 expresson_help

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Posted 02 February 2005 - 06:55 AM

Recently when I was doing SDM I used Promega's GeneEditor system. That worked well for a single base mutation.

#5 labprincess

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Posted 03 February 2005 - 06:07 PM

You could try to up the amount of primers in the reaction. Yes, a good part are gonna form dimers (that's just life :D ), but with the increased primers you should get some more binding to template DNA . I have found the bumping up the template and the primers seem to work (however with the increased templete, background colony numbers can go up unless you are careful in getting rid of template plasmids) oh yeah...I use HPLC purified primers


I hope that helps :)


LabPrincess




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