My plasmid for site-directed mutagenesis id loosing my gene of interest. Is there any way to dill with this problem?
Check your cloning conditions, your plasmid DNA. You may want to try using a different cloning vector or different method of mutagenesis.
Hi Ilkona,
1- are you sure of the starting plasmid? (make some restriction digest/sequencing)..is it pure? (ie the deletion is already present in your DNA prep...maybe a good idea to isolate a new clone...
2- do you have repeated sequences in the gene/ plasmid you mutagenesed or in the oligo you used? polymerase can jump from one repeat to the other and if a deletion product is made it will be amplified more easily and overtake the full length product
3- for the same reason, make less PCR cycles; if it is a point mutation you want to introduce, 12 PCR cycles should be enough (with more cycle if a deletion product is made it will be amplified more easily and overtake the full length product)
good luck for your experiment
Did you streak to single colonies? A mixed culture will favor the replication of the smaller plasmid & can outcompete bacteria with the larger plasmid (the one with your gene of interest).
Are you maintaining your bacterial cultures under selection?
Lots of possible ways for this to go wrong. And as Didier Poncet pointed out already, are you certain that you have the correct plasmid?
- Badges
- Science topic