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Finding the right sequence - (Feb/16/2012 )

I've been having trouble with RT-PCR and have finally ruled out everything except the primers. I ordered control RNA and control primers and got the expected band there, and I'm fairly confident I have good RNA (nice bright 28S and 18S bands), so I'm pretty sure the problem is my primers.

My goal is to confirm the results of the genome microarray experiments that were done by someone else. This is for rat spinal cord RNA expression.

To start I selected an internal standard, based on the literature I picked a ribosomal RNA, Rpl27. I used genbank to identify the sequence and came up with NM_022514. I put that into PrimerQuest (primer design tool available on Integrated DNA Technologies website). That gave me 5 primer sets and I ordered all 5 and none of them gave me a band.

Any suggestions?

-Rachel Ruhlen-

Well none of them gave you a band... did you try to optimise the reaction? What annealing temperature you used?
The gene is not GC rich, and the primers look fine. Ta 60 should work.


I used 60C for the annealing temperature.
I tried adding a ul of Mg to the buffer.

-Rachel Ruhlen-

so titrate in the mag chloride


I strongly suggest trying a 55C annealing temperature. It mostly works.