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Fluffy

Member Since 18 May 2011
Offline Last Active Feb 20 2013 01:42 PM
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#112568 Please help! no PCR bands+Description of my PCR protocol provided :(

Posted Fluffy on 14 June 2011 - 01:03 PM

View Postphage434, on 12 June 2011 - 07:27 AM, said:

Your primers have RE sites at the 5' end, but are missing the required 5' overhang needed to allow you to cut your final product.  This should not affect your PCR reaction, so you can optimize it using your existing primers, but you probably should order new primers, which will be needed for your next step.  I'd recommend at least 4 bp of 5' overhang.

Hello Phage 434
Yea I will look into that. I believe you are right. My professor also talked about this issue and hopefully will be solved.
Thanks Phage :D:D


#112567 Please help! no PCR bands+Description of my PCR protocol provided :(

Posted Fluffy on 14 June 2011 - 01:01 PM

View PostGNANA, on 11 June 2011 - 11:43 PM, said:

Analysing your primers it doesnt seem to be of the tm range you have mentioned, it is far lesser than that...to my prediction it is around 55 degrees only (Excluding the overhang RE sites)...so i think your PCR should work fine if you set your Annealing accordingly, may be i would set between 53 and 55 as annealing for this tm...

Good luck...

Thanks Gnana for your feedback. When I ordered my pimers, the Tm written on their tubes was in that range :$. But I will definately try that annealing range you mentioned.
:D


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