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degenerated primer for nested RT PCR


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5 replies to this topic

#1 teerachild

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Posted 10 January 2002 - 11:27 AM

Dear all, I'm a master degree student. This is my first time to do nested RT-PCR and I made primers for PCR already but I'm not sure that the primers will work coz they are degenerated primers. Well, I asked my advisor and he said it's OK. but I think the problem would occur coz they are high different in Tm for each possible primers. Please email me , teerachild@yahoo.com
Thank you.

#2 Leong Wai Fook

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Posted 17 January 2002 - 02:51 AM

It's OK especially for the RT part since the temp for it is low. But for PCR, you have to compromise the Tm for the one of the lowest. Usually choosing a lowest tm for degenerate primers works. For my case, I have Tm as low as 45C and it works. Just try and see.

#3 teerachild

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Posted 17 January 2002 - 04:49 AM

Thank you. I'll try.

#4 Leong Wai Fook

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Posted 19 January 2002 - 02:19 AM

Good luck

#5 teerachild

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Posted 22 January 2002 - 12:01 AM

But if the company sent me primer pool in one tube and there's only one annealing temp, 46 c. Should I lower the temp or just use it?

#6 moreyroger

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Posted 29 January 2002 - 07:48 AM

Just use it.  

If it doesnt work, try touchdown from 51-43C.





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