2 replies to this topic

[jaimev]

So I have been trying to optimize PCR conditions for the last few months and have minimal experience with PCR. Additionally, we don't have anyone in lab with experience so I apologize if this is a basic question. I recently discovered a problem after having several successful PCR optimization results. I use PCR to look at 5-HT1A receptor levels in rat brains, but wanted to start off showing knockdown in cells (this is just regular RT-PCR not qRT-PCR). Prior to recently, all of my controls have been working just fine. I then suddenly started getting bands in my negative control RIGHT at the level of my band of interest (500bp so probably not primer dimers). So I used fresh aliquots of everything and still a faint band. I checked my loading of the gel by placing the negative control by itself, but still a faint band. Then I tried using new water from another lab with no luck. Finally, I then tried running two negative controls; one in which there was water and primers with mastermix and also one that had only water and mastermix but no primers. The sample without primers had no band while the primer negative control did! Interestingly enough, my untransfected cells (which had primers) also don't have this band. So if it was linked to my primers, wouldn't I see it in my untransfected cells? I have a picture I can also try uploading later, but basically I was wondering if anyone knows what I would get amplification in my negative control with primers, but not with untransfected cells (which had primers) and a negative control without primers!? Any information would be greatly appreciated. Trying to illustrate knockdown of this receptor will be the death of me!


Ok. So I loaded the picture and I know that I have some non-specific amplification higher up (which is also new), but I don't want to get into that at the moment. The last band in the ladder is 500bps which is where my band of interest is located. The order of my samples is as follows:

1) ladder
2) untransfected cells
3-7) transfected cells with my receptor
8) negative control with primers
9) negative control without primers
10) positive control with plasmid DNA

[pcrman]

There is no doubt that your PCR system is contaminated. You said you replaced many reagents, but have you also ordered new primers yet?

Also be aware that contamination may not only come from reagents, but also from your work bench, pipettes etc. You should clean up those things and assemble your PCR in a designed area just for PCR. Using aerosol resistant tips is also important to get rid of contamination.

[jaimev]

pcrman, on 31 July 2012 - 05:48 PM, said:

There is no doubt that your PCR system is contaminated. You said you replaced many reagents, but have you also ordered new primers yet?

Also be aware that contamination may not only come from reagents, but also from your work bench, pipettes etc. You should clean up those things and assemble your PCR in a designed area just for PCR. Using aerosol resistant tips is also important to get rid of contamination.

I made fresh aliquots from original stocks so I guess my stock could be contaminated, but then why no band in my untransfected cell? I keep an area designated for PCR and wipe it down, wipe pippettes down, use special tips etc.  And why does it run exactly where my other bands do?