Annealing temperatures for long primers? - PCR reaction (Mar/15/2006 )
Just joined and hope everyone can help!
Trying to do a PCR cloning reaction but have several problems with the 2 primers I need to use:
- they are quite long - 43 and 30 ntds but 13 of the ntds are restriction site sequences added at 5' end and don't match with my template
- plus one of them has a GC content of 67%
The Tms are listed as 75 and 75.2.
What annealing temperature should I be using? The above Tms or should I recalculate using only the sequence that matches to my template?
So far have had no PCR product - tried adding 2% DMSO but only saw high weight non-specific product.
What are you using such long primers for, can you be bit elaborative, I may be able to help.
for this stuff, i use the tm of the matching part of the primer for the 15first round of PCR, and increase the anneal temp to the maximum i can (mean 68 for Taq (72°) elong°, or in general (elongation time - 5°) for 25 further cycles
You calculate the tm of your primers with the part that anneals to your template. It generally works well. Normally you try to design similar primers in terms of tm (similar tm of the part that anneals). However yours may be quite diferent (43 and 30)?
I went away and tried your suggestions and in the end I got my PCR product using fred_33's suggestin of 15 cycles at 1 temperature and then more cycles at a second temperature.
Problem was I had a string of A's and T's in my sequence and had to extend my primer long enoug so I could get a decent enough melting temperature and GC content.
But you forgt to thanks the enzymes, and the small bacterias used for enzyme production and..................
ha ha oh yeah.. thanks to all the little people.. er little bugs!