1.5kb cDNA segment won't amplify

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1.5kb cDNA segment won't amplify

Postby fmorton » Feb 05 2017 3:50 pm

Hi,

I'm trying to amplify a 1.5kb segment of the hepatitis c genome. I set the thermal cycler to 35 cycles and changed the annealing temperature to 60degrees and increased the extension time to 1 minute and 30 seconds, I didn't see any amplification when i ran my gel. I was wondering is there anything i could change to get amplifcation? maybe increase the number of cycles? I'm an undergrad student so I'm not a pro at PCR (yet)
thanks!
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Re: 1.5kb cDNA segment won't amplify

Postby mdfenko » Feb 06 2017 12:15 pm

why did you set the annealing temperature to 60C?

the rule of thumb is to set annealing at 5C below the melting temperature of the lowest melting temperature primer (although the primers should have approximately the same melting temperature).

give us more information about your protocol.
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