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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:

pcr product:low specificity\high specificity\higher specificity.

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



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.


QUOTE (Madrius @ Jan 21 2008, 07:30 AM)
sorry,I made a mistake.The specificity is just opposite.


pcr product:higher specificity\high specificity\low specificity.

Thanks for your reply~~


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.