I think it might be the fact that I put it at -20degree Celcius after the reaction. Can it be a cause of DNA degradation, should I keep it at 4degree Celcius instead?
Storing at -20 degree Celsius should not be an issue, in fact that's what I usually do if I do not transform the same day. What kit are you using, Mayerline?
I use the QuickChange lightning site-directed mutagenesis from Aligent and the length of my primers are 34nt.
I follow exactly their protocole and it still doesn't work.
The kit you chose is very efficient. Make sure the concentrations of the primers and DNA you are using are at the right concentrations. Double check that your primer sequences are correct as well (this is a common area for error). Also, for the cycling parameters, the step time length at 68 degrees Celsius is correct depending upon your plasmid length (30 sec/ kb of plasmid length). For transformation of XL10-Gold cells, I normally use 10 ul and add 0.75 ul of DPN-treated DNA to the cells.
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