3 replies to this topic

[SpiralX]

Hi,
I am doing PCR to optimize my primers which I'll be using for qRT-PCR, but I've been getting bands in my -RT as well as negative control. I changed everything possible,
1)right from my pipettes (which are relatively new),
2) PCR tubes,
3) reagents,
4) water
5)we even ordered new primers.
After changing the pipettes though there wasn't any band in the negative control only -RT, but when I repeated it the bands have appeared again. I have no idea what do I do? I must also mention that the RNa extracted is form THP-1 cell line using nucleospin, during which DNAse are used so there is very litlle chance of genomic DNA contamination.

[Nephrite]

Hi. What kind of bands do you have - primer dimers or a real amplification product?

I had a similar problem but my primers were cDNA specific so I did not run ''-RT'' controls. I didn`t know anything in this area and somebody in the lab decided that I have been contaminated my primers with DNA from the environment. Therefore, I run three NTC containing both primers, only the R and only the F primer. I had the 50 bp product only in the NTC with the two primers, i.e. the band was a cross-dimer. I thought that without getting rid of the cross-dimers observed on the gel it is not possible to do the qRT-PCR. I shared my problem and one of the more competent users here advised me ''Just do the real time-PCR and see whether you have a problem to worry about'' - the best advice I have ever had so far :-)

Hope this will help you.

[SpiralX]

Well I've been getting actual product amplification. My product is 134 bp and i am getting similar bands when I run the -RT and NTC, indicating that something has been contaminated with genomic DNA and i am having a tough time finding what it is..and unless i sought this problem out, i don't think i'll be allowed to proceed to do  my qRT-PCR.

[Trof]

SpiralX, do you use sterilized filter tips? If not, crosscontamination through the pipette is possible.

Also, not all DNases work well, colleague told me that for example QIAGEN on-column DNase treatment did not remove all the DNA traces and has a RT- positivity. Are your primers intron spanning (or placed on exon-exon junction)? Did you tested them on DNA?