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Dear all,
I am trying to clone a gene of about 2kb into a plasmid (TOPOV5/TA cloning), after cloning and transformation into competent Ecoli(selecting via blue/whit screening) I tried to confirm by amplefiyng an area flanking the end of the plasmid and the begining of the gene, so i got the product and it was confirmed by sequencing, after that i tried to introduce a mutation by site directed mutagenesis (round the horn) but it failed, note: when i try site directed mutagenesis (SDM) using commercially obtained clones that contain with the same kit and condition and primers it works!
my questions are
1) what could be the problem with my hybrid clones? if it contains the insert can ther be any other problem like confermation of the plasmid or extraction method?
2) is what i have done so far enough to ensure proper insertion of gene into plasmid?
thank you very much
