information regarding PCR using very long primers - (May/07/2007 )
i want to amplify a gene that has primer seq. of 53(F) and 55® bases.the primers have GC content of 37-40%. but their tm is 82.im using annealing of 55 for 1 min and im still not getting any bands.pls suggest.
try different annealing temperatures, go higher like 65
Try using more DNA template. like 100ng
im actually not getting any amplification , just primer dimer. thereby i believe increasing annealing temperature would mean increased specificity and thereby lesser chances that such a long primer would anneal to the template. other than 55, i have used 50 also as the annealing temperature but still i cant get any band other than primer dimer. i have changed my enzyme(taq), dNTPs as well as MgCl2 to eliminate the thought that there could be anything wrong with them.
i have triend 4 different template and primer concentrations too:
template:500ng, 800ng, 1000ng and 1200 ng in a reaction of 50 micro liter.
primer: 5pm/microliter, 10pm/microliter,15pm/microliter and 20pm/microliter for 50 microliter of reaction mix.
i have even used DMSO at: .1%, .2% and .25%
i am currently using dNTPmix with 200micromole conentration each for 50 microliter.
and MgCl2 at 1.5mM
please suggest what should i do.
Play with gradient and concentration of MgCl2. It might help. Increase the concentration. Sometimes long primers are pretty tough to get band.
as timjim suggested, try higher Mg conc. try 2& 4 mM
check the integrity of the template DNA on a gel.