how can I shift the frame? - please help!!!! (Nov/01/2006 )
i need to add 2 bps or to remove 1. i am not sure what is your question? 
-Kathy-
Oops sorry... there has been a misunderstanding on my part.
-perneseblue-
QUOTE (Kathy @ Nov 6 2006, 01:32 AM)
Missele thanx a lot for the advice. the thing is that i never used mutagenesis so im a bot worried about it.
what do you mean by that? how can DpnI digest if i dont have its site in the DNA? and why to do that?
QUOTE
then digest the parental DNA with DpnI (I used 1 µL that is 10 u) 1hour 37°C, and transform bacteria with 1 µL.
good luck
good luck
what do you mean by that? how can DpnI digest if i dont have its site in the DNA?
and why to do that?DpnI will digest the parental DNA because it has methylated bases, therefore it will not digest the amplified DNA. It's better to digest the parental DNA to get rid of the non mutated plasmid and to increase the chance to transform your bacteria with mutated plasmid. But you still need to sequence the resulting plasmid because sometime you don't get the mutated one.
-Missele-
dpnI digests the sequence GATC when methylated. I would be very surprised if your plasmid does not have that sequence a single time...
-vairus-
i see. thank you very much! 
-Kathy-