I have designed primers to isolate a promoter from rice.I used the Life Technologies OligoPerfect™ Designer.
I obtained the following primers:
1) Forward Primer : 5'-T T T T G A A T G C T G G A A T G A T A A G C-3'
2) Reverse Primer : 5'-C A C T G A A G A T A T A A T C T A A C T A G C T A G C T A -3'
On analyzing the reverse primer in IDT oligoanalyzer, I found this in self dimer analysis
On using 5' overhangs containing RE sites for cloning , in the reverse primer the IDT Oligoanalyzer showed this during self dimer analysis:
Now what should I do if I decrease the length of the reverse primer, the Tm difference between the primer pairs increase more than 5 C.What should I do?
I know the formation of primer dimers is inevitable.
Is it possible that I first preheat the primer and water at 95 C for 5-7 mins and then add the rest from the master mix.Will it affect the Taq polymerase?
I cant afford to buy Hot Start Taq polymerase now.Can I do the same thing with regular Taq?
What should I do? Please help.
I havent ordered the primers yet..