cloning a gene in to a plasmid - (Oct/08/2012 )

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

Ideally you would have sequenced the full insert using the primers for the plasmid you are cloning into, these are likely to be M13 universal primers or something like BGHpolyA reverse and something else for the fwd.

SDM is difficult - the size of the plasmid alters how long an extension time you need and how efficient the reaction is. Try making the extension time longer and perhaps fiddling a bit with the Mg concentration in the buffer.

Are you doing the Dpn1 digest at the end?

Thank you very much bob1..and yes my plasmid that i use is 5.5KB with out the insert but the commercial one is 3.5 together with the insert!
and i use DPN1 after SDM it works perfectly with the commercial plasmid , where usually a sharp band of the same size of plasmid sti exsists after DPNI treatment, but with my insert i get many faint bands that simply disapear completly after DPN1 treatment...!
ill try your suggestion it sounds very logical thank you

Hello,
I have an other question regarding transfection, I transfected HEK293 cells with plasmid that carries my gene, and I selected cells that are stabelly expressing my gene by antibiotic selection, the protein that I am interested in studying should be execreted in the media of the cells , so in order to be sure that I have my protein I preformed a westren blot using a MYC antibody as my protein was myc tagged and i used the media of my protein directly , the problem is that every time i do a westrn blot i get the same pattern of bands even in control sample that was not transfected,one of the bands is in the right size place but i dont trust this result,i tried using my protein antibody i get the same result? any idea about what is happning?

You may need to optimize the antibody for the system. You could also try seeing if the cells are secreting the protein by doing immunofluorescence on the cells (with protein Ab and myc if necessary), you could also try an ELISA for the protein, which should be more sensitive than western blotting. If the protein isn't normally found in 293's you could also look for RNA expression to confirm that the plasmid is inserted and working.

Note that selection ican a bit of a problem - this is usually where these sorts of things fail. You may be better off starting with transient transfections to ensure that the whole system works before going to stable lines.

the protein is narmally expressed in the hek cells but not the myc tagged ones, i will try the ELISA and see.
thank you very much