1.5kb cDNA segment won't amplify

Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies

Moderators: r.rosati, mchlbrmn

1.5kb cDNA segment won't amplify

Postby fmorton » Feb 05 2017 3:50 pm


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)
Posts: 2
Joined: Feb 05 2017 3:41 pm

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.
talent does what it can
genius does what it must
i do what i get paid to do
Posts: 1476
Joined: Jan 24 2006 7:39 pm
Location: Staten Island, NY USA

Return to DNA and General PCR Methods

Who is online

Users browsing this forum: Google [Bot] and 7 guests