double digestion and ligation - (Jun/17/2008 )
these days am doing D.D with EcoRI and BamHI for pMV261 and pcDNAT4/TO; to insert 1017bp of PCR product and I cut one after one and i checked after every step for bothDNA and Plasmid and I get good result with one enzyme cut for plasmid and also with the second but for the pcr product when I used the bamHI Igot good result but with ecorI the result appeared smearing in the gel , and also some time when it give me good result with the PCR product in D:D but still I have problem with ligation reaction no colony so if any body have idea what can ido thanks for any suggestion
In principle it should be possible to cut with EcoRI and BamHI in one reaction mix. However I had also some problems with EcoRI/BamHI digestion of PCR products. I overcame it with first cloning the PCR product via AT-cloning into pGEM-Teasy and from there I did a subcloning into the target vector. How many nt are flanking the digestion site of the PCR product? If there are only 2 or 3 bp this may explain your troubles. Some enzymes need 5 or more bp to cut properly.
yeb, firstly I did this mistake with my forward primer and NOW I have 2 primer one with 6 flanking bp and other with 15 bp, by the way did u know the best programme to check the designed primer bcs many programmes give false results.and did u used the same RE(ecori+bamHI) for subcloning.
I guess I'm a bit old fashioned in things of primer design don't use any design programs. I take a sequnce left and right of my sequence of interest (about 20 bp)and add the digestion site (look if the Tm is ok -> 4+2 rule and there is a G or C at the ends). Since I'm cloning via pGEM T easy I don't care about how many bp are prior my digestion site. For the subcloning I'm using the inserted digestion sites.
By the way, did you check if your ligation and transformation method is working? e.g. Ligase Buffer OK -> maybe ATP precipitated? If you're doing electroporation: time constant Ok? Are the cells competent enough?