quick change mutagenesis...... - (Apr/20/2013 )
Hi Gnana...As per ur suggestions i prepared Top10 competent cells and i got colonies in both DH5alpha and XL1 blue competent cells....I also screened for the plasmids and the plasmids are also fine....further im also trying to verify using Restriction digestion and PCR...So give me an idea how many plasmids shall i send for sequencing? both from XL1 and DH5alpha? Thanks in advance
To start with i would sequence 3 plasmids. mostly i ll get all three positive for mutation, then i choose 1 or 2 from it and sequence the entire insert to make sure there is no other PCR induced variations.
Gnana my gene was cloned in Xho1 & EcoR1 sites....So i took some plasmids from XL1 & DH5alpha transformed after following mutation protocol....But i could see out of them only one plasmid has released the insert...My positive control also have worked....What would be the reason? Shall i give the ones which have released the inserts after digestion 4 sequencing? I am also doing PCR with those plasmids...Kindly give me your valuable suggestions....
Actually there shd'nt be a probelm with the presence of insert, but make sure the improper digestion is'nt the cause for that. anyways you also checking them with PCR. personally i prefer restriction digestion than PCR, as the possibility of false positivies/negatives are more with PCR screening. however, with proper controls you can interpret that.
your positive controls that worked for digestion is a similar prep (miniprep/maxi or plasmid isolation protocol) like the mutated preps??
sometimes PCR induced variations may abolish the RE, but i believe you would hav used high fidelity taq or PCR mix that could be used for mutagenesis.
if not the above, make sure your cells are sterile. some times you ll get colonies due to contaminaton... but following transformation procedure without adding plasmid or just water could help you to check the sterility of the cells.
since you are having the insert issues in the fellow preps, double check the plasmids that released the insert are specific by digesting with other RE's, and then i think you can send them for sequencing, dnt forget to sequence a wild-type plasmid along.
No the positive control is the maxi prep plasmid isolated from kit...But i screened the plasmids manually...Also i used Pfu Turbo DNA polymerase only...While isolating plasmids manually i did not give 70 percent ethanol wash...Will that be the reason?Im sure my competent cells are sterile...While running plasmids after isolation all plasmids showed same retardion compared to my empty vector. Will take your suggestions Gnana....Thanks
so no issues with the taq.......so the purity of your prep might be the possible concern here. so reprecipitate your plasmid and digest again, or go with miniprep kits for isolation and proceed.
about the retardation on gel, not always you see the difference between the empty and insert containing plasmid, so need'nt worry abt that.