RT-PCR and Negative Controls&Contamination

ddam at uh.edu ddam at uh.edu
Wed Jun 2 21:41:42 EST 1999


Help!! Anyone...(rather long)

We are performing a standard curve quantitative RT-PCR assay with varying
results. I had my students run negative controls:
1. no RNA template in the RT reaction
2. no template derived from the RT reaction in the PCR reaction.

Results: We see bands at the proper bp length of our Target wildtype and
competitor DNA (ie. the size we engineered if we had added competitive
RNA in the RT reaction). WITH NO RNA INPUT.

Therefore we decided to change everything: New Depc H20 (Ambion and in
house), new Oligo dT , new primer sets (all using Ambion water), new
reverse transcriptase enzyme, dtt and buffer; new HotStar Taq master mix
etc.

Aerosal tips have been used along with RNase, DNase free tubes, tips etc.
(We haven't tried DNAzapping the thermocycler (MJ 200 with hot bonnet
lid).

I am at a loss! We had another lab run our 2 primers in a PCR reaction
and they found 1 or both bands too, suggesting that the new oligo stock
is contaminated again. The primers are to a 228 bp region of a conserved
region of pro-alpha I type I collagen. When the oligos are added in the
PCR reaction, they generate bp DNA fragments of 228 and 188 (just like as
if we had put RT derived cDNA template in the PCR reaction).

Does anybody have any ideas what we can try next? It is throwing our
standard curve off big time! Log ratio 0.78!

At least when we put no oligo's or a non-homologous primer pair in the
reaction, no bands appear!

Thanks in advance,

Dan-O


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