Mutation - (Jan/08/2007 )
Hello every one
i got problem i have vector with 2 Bamh1 site i need to deleted one of the is there any process to do it or kit
Thank you a lot
you could do a partial digestion with BamHI. which will require you to first find out the amount of enzyme to add and the amount of time the digestion need to run. You can do this by either setting the restriction enzyme constant and vary the time. or setting the time constant and varying the amount of enzyme added. Partial digest is stopped usually by EDTA.
-only you have have the partial digest.
-gel purify the partial digest,
-excising the linearised single cut plasmid,
-extract the DNA,
-full fill in the overhangs with Klenow or add in a linker.
-ligate, then place in e coli cells
-then screen for a plasmid which has the correct BamHI site deleted
How abt site directed mutagensis kit. May b this would work.
i did the digestion with partial digestion with BamHI but i didn't success .
Wrong bamHI site deleted? Or there were no colonies.. ie the ligation failed.
Wrong bamHI site deleted
how many colonies did where checked?
I would check around 48 colonies. If the partial digest worked, then it is only a matter of screening sufficient colonies to find the correct clone,
i got around 12 colonies