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Tm caculation for different PCRs?? - new, confused, and frustrated beginner (May/26/2010 )

Hi all,
I am confused and exausted about PCRs... PCR, RT-PCR and mutagenesis PCR...
Any more?
For the Tm of primers, they have different ways to caculate? Even for the annealing, elongation temperarture. Despearted asking for help :P

Now, my current headache. To desigh a site-directed mutagenesis primer for a high GC% region. I did design as the protocl suggested; 1) Tm>78C, GC at the both ends, and proper size.

Then, my nightmare came. Because of the high Tm, and GC rich, (or something else), I got nothing after DpnI digestion. Then, raise the annealing temperature to 65. And 68C is the elongation temperature suggested by the kit. Still nothing.... What the hell!!

Well, can some one give a general diagnose and small "lecture"!
Thanks a lot :P

-CAT-

i do not know much abt mutagenesis PCR. but have u heard about two step pcr? where annealing and extension/elongation can be carried out on same temperature. i wonder if this can be useful.
as for getting no results, this can be due to many many other reasons, other than the annealing temperature problem. in my opinion, you need to sit down again and review ur cocktail recipie. u can increase primer conc, u can increase annealing time, u can increase elongation time. therz much u can do. and lastly, getting the results of pcr for a beginner is never easy, so keep trying and dont get frustrated by just two failed experiments. prepare urself for much larger number of failures in PCR :)

-Muhammad Umer-

Muhammad Umer on May 26 2010, 11:38 PM said:

i do not know much abt mutagenesis PCR. but have u heard about two step pcr? where annealing and extension/elongation can be carried out on same temperature. i wonder if this can be useful.
as for getting no results, this can be due to many many other reasons, other than the annealing temperature problem. in my opinion, you need to sit down again and review ur cocktail recipie. u can increase primer conc, u can increase annealing time, u can increase elongation time. therz much u can do. and lastly, getting the results of pcr for a beginner is never easy, so keep trying and dont get frustrated by just two failed experiments. prepare urself for much larger number of failures in PCR ;)


Thank you, Muhammad! Two step PCR? Mm. I am going to dig that out!
Thanks!

-CAT-

I'm not sure if this applies to site-directed mutagenesis too, but PCR with high GC content usualy doesn't work without additives like DMSO (2 - 10%), formamide (2-10%) or betaine (0.1252.0 M), because of formation of secondary structures.

-Trof-