reducing primer dimer - (Jun/23/2005 )
I am new here and what to have suggestions for reducing the primer dimer.
I am doing the PCR using cDNA as templete. The primer consists gene specific and linker which make the primer 60 mer long. The PCR worked fine when using the gene specif primer. The dimer appeared when the primer is fulllength (gene specifi plus linker). I use the Expand PCR system touch down. I have tried other PCR enzymes and different conditions such as primer concentration, cycles, annealing temp, and templete but seemed not much helpful. Could you please give some ideas about the dimer. You kind help is apppreciated.
The standard lines on reducing primer dimer are:
1/ Increase annealing temperature - presumably the primers won't have as high Tm as the specific target.
2/ Re-design primers to avoid complementary sequences.
3/ Decrease the primer concentration as this would decrease the excess primer available.
4/ Other suggestions include the addition of additives
Hope the helps