What's wrong with my pcr? - high temperture with low specificity (Jan/21/2008 )

[font="Impact"][/font]hi! everyone~~~~
There is a problem with my pcr amplification!I hope somebody could help me out.

My pcr template is plasmid.

condition and results as follows:

temperture:55\58\61
pcr product:low specificity\high specificity\higher specificity.

what's the matter?
I hope somebody can give me a explain.
Many thanks!


youngbing

I can't really understand your problem...

If at 58ºC (or 61...) you get a high specificity for your amplification, what else do you need?

Maybe I got things wrong...

What is the problem?

I'm not sure I'm getting this neither...

But one thing that seems ok, is that a higher temperature gives you a more specific reaction. This is caused by a greater speciticity of your primer for your template.

have you changed any other parameters such as buffer amount or more specifically speaking; amount of MgCl2. if you increase amount of MgCl2 stringincy (specificity) decreases and if you decrease MgCl2 amount under a certain level Taq po. can't work properly.

Thanks for your reply~
I'll change parameters and try again.

is it primer OK?
i mean, is it going to give you any binding else where?

primer design software just give you a guideline... sometimes, might not be accurate...
if really not working, try design another pair of primer and see.