Enzyme sites on Primer - (Sep/19/2007 )
I want to ask you that does Kpn1 site on forward primer and Xba1 Site on reverse primer can be used. Actually i am little bit confused in overhangs produced by these enzymes. Does it matter or not or there is some specificity according to overhang.
Xba makes T^C T A G A
Kpn makes G G T A C^C
there is no pb to use them. no possibility of cross reacting.
also check the page "restriction close to dna ends" from NEB to check additional bases needed for these sites top cut properly
I have always had trouble with cloning using RE sites in primers. I have had a lot more success cloning the PCR product into a general PCR vector like pGEM-T and then using the RE to cut out the insert and then subcloning into the vector you want. Even though this seems longer it seems to be much more reliable than fiddling around trying to clone directly by RE digestion of the PCR product.
PeakTrace ABI Trace Basecaller
well i don't true agree as digesting pcr fragmetn avoid the gel purification step which causes me many many problems.