help with RT-PCR - DNase usage - (May/13/2008 )
i am finding trouble with my RTPCR. I am trying to find the 5' end of mRNA of a protein. I isolated total RNA and did RT (two reactions, one with gene specific reverse primer- from the known portion of the gene and another with oligo dt). I used this cDNA from RT to ligate to an adaptor molecule and PCR with gene specific reverse primer and primer complimentary to the adaptor oligo. I got very specific band, when i sequenced it, i did not find the 5' end of the mRNA...i mean i could find a continous ORF or part of the known protion of the gene. It seemed my RNA isolation was okay (i used QIAGEN columns), RT reaction is okay since RTPCR gave band ( i tried using different gene specific primers too) also my ligation with adaptor oligo has worked since i have got the adaptor sequence in the PCR product. This was going on...i was getting frustrated since i wasnot getting any gene specific sequences though all my techniques are working. Then one day instead of loading my PCR product, i mistakenly loaded my cDNA sample in the agarose gel and found that there was a very huge band of size more than 12 kb, i am suspecting that it is chromosomal DNA...because cDNA being single stranded would not appear in the gel and also it would not be that long (more than 12 KB). so all these PCR bands that i am getting...are they all from the Chromosomal DNA? is my adaptor oligo binding to chromosomal DNA..is that the reason for me to sequence that doesnot have a continuous ORF ?
if i use DNase..while preparing RNA...will that help?
please help me
thanks in advance
Qiagen has a Dnease digestion in the column for the Rneasy protocol.
Ambion's Turbo DNAfree kit is much better than qiagens