5' RACE cDNA Concentration After SNAP Purification? - (Mar/21/2012 )
Hi, I'm trying to do a 5' RACE but I'm not getting a clear product. It is very smeary and the amplicon is not even the expected size. I am using the "5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0" from Invitrogen. Primers (GSP1 and GSP2) have been tested on genomic DNA and work properly. I suspect that my template cDNA concentration might be too low.
After SNAP purifying the reverse transcribed cDNA, I nanodropped my sample and it showed 5-8ng/ul, which seems very low. Perhaps the transcript I'm trying to amplify is not expressed at high levels? Does anyone know what the template cDNA concentration of a sample before PCR should be in a successful 5' RACE reaction?
I am also running 5' RACE but I keep failing and get bands that are just self-ligated anchor primers at different sizes. However, I'm using T4 RNA ligase which must be different from your Invitrogen kit (most of commercial kits use terminal transferase). I hear many people get smear during 5' RACE with commercial kits (many of them used Invitrogen). If you read other posts on this forum and search for 5' RACE you must find the same issue. I think 5-8 ng/ul is still ok, and you must see something with it, but it depends on the purity of cDNA too. next time add as much as you can, just don't exceed 10% of the final volume of PCR reaction.
primers for genomic dna may not be suitable for cdna. they may be specific to intron sequence or cross intron/exon borders. they must be completely within the exon (one that is part of the cdna, watch out for alternative splicing) to be useful with cdna.
This is a bacterial genome so I don't think there are any introns. Also, it is a fairly well defined DNA sequence and I doubt that my professor would have overlooked errors in the design of my primers. Because of the cDNA concentration I obtained from the nanodrop, I really think it has something to do with the concentration of the target transcript or its rate of reverse transcription or rate of tailing.