Methods for checking DNA Contamination - (Feb/22/2007 )
I am using the Taqman RT-PCR method and am having trouble in working out the best way to check for DNA contamination in my samples after first strand reverse transcription.
Isolated RNA is checked on a nano-drop and run on a gel to check for dedgradation, it is then treated with the Ambion Turbo DNA kit befroe reverse transcription. A lab memeber then uses SYBR green to check for gDNA contamination. Is this is the usual method? Would I expect to see no signal in the -enzyme sample if there is no contamination. Also why does she use primers? surely if there is contamination then you would get a signal?
Sorry for all the questions! I really hope someone can help as my head is about to explode!
no, you need primers. you might get a signal if there was a whopping ton of DNA contamination, but if your amount is very small you won't see it till you amplify..but it's still plenty to mess up your numbers
SYBR green is a good way to check, if you are running melting curves. this shows you the difference between amplicons. also, even with a primer-probe system, wherever possible use intron-spanning primers; this makes it much easier to rule out DNA contamination
does that help?