LacZ PCR genotyping -ve control contamination - (Feb/13/2010 )
Hello everyone. I am new to this forum and wonder if someone could help me with a rather annoying problem as I have tried to troubleshoot it the best I can!!
I have been getting a band popping up in my negative controls in my genotyping for the lacZ transgene. The contaminant band is the same size as that of the LacZ (~315bp)
I used fresh PCR reagents, fresh dilutions of primers, water, pipette tips etc. and I am still getting this contamination. I have done this several times now.
I have ordered new primers as I suspect the contamination might be there.
If anyone has any experience of having this problem, particularly with lacZ (which I am told is rather susceptible to contamination) I will be much appreciated if you could help me out!!!
You are working off a plasmid that has probably been extracted thousands of times in the lab you are working in, I'm not surprised you have contamination issues, the air will be full of it.
I suggest trying setting your PCR up in another room if possible; somewhere where no plasmids are extracted/bugs grown. If that isn't possible, try keeping a set of pipettes just for setting up PCR, and have a PCR region, where you only set up PCRs.
bob1 on Feb 14 2010, 03:30 PM said:
I suggest trying setting your PCR up in another room if possible; somewhere where no plasmids are extracted/bugs grown. If that isn't possible, try keeping a set of pipettes just for setting up PCR, and have a PCR region, where you only set up PCRs.
I set up PCR in another room as you suggested and I am still getting this contamination occurring. I even cleaned my PCR workstation quite thoroughly and it didn't make any difference. I used fresh primers that arrived today and I still got the contamination.
I wonder if DMSO might be causing this (although I have used two different bottles to check if that is the cause of the contamination, no luck)
Maybe your pipettes are contaminated. Are you using barrier tips?