contamination in cDNA - (Mar/02/2007 )
It looks like I have genomic DNA contaminating my cDNA. IS there a way to clean this up ( Iam using SYBR Green as my detector).
nope you have to make sure it is not there in the first place (in the RNA) otherwise you will digest the cDNA too (because it is all DNA) To remove you do DNAse treatment on the RNA before reverse transcription.
I did a DNase treatment step, as I always do. Usually the samples are clean. How do you check for DNA contamination before the qRT run?
use an aliquot of the DNAse treated RNA in a PCR reaction... The RNA will not amplify the DNA should... best is to use an amount equivalent to the amount of RNA you will use in each PCR... ie: I am making a 10ul RT with 500ng RNA I will use 1ul of the cDNA for PCR so test for DNA contamination on 50ng RNA... this is the same as the -RT control but can be done without doing RT first...
Thanks much for your help!