Contamination Problems! - (Jul/19/2008 )
i've been having contamination problems with my PCR. a distinct band is formed in the negative controls and the contamination is of the same size as my product. but the reagents have all been changed except Taq polymerase, and master mix preparation and template addition is done in 2 different hood, while reagents and template are kept in different refrigerators.
i also face problems with my DNA ladder. the larger bands of the DNA ladder are unable to resolve properly, resulting in a smear. the gel run was performed at 100V, 400mA for approximately 70 mins in a 1.5% agarose gel (TBE buffer)
any suggestions ?
i also face problems with my DNA ladder. the larger bands of the DNA ladder are unable to resolve properly, resulting in a smear. the gel run was performed at 100V, 400mA for approximately 70 mins in a 1.5% agarose gel (TBE buffer)
any suggestions ?
Hi! I faced the same problem than you. In some cases I realized the end of the comb was very close to the casting try and a sample was contaminatng the next one from the bottom.
Good luck! Angela
could it be an accident when you load your gel... sample from a well accidentally flow to another well next to it.
or, maybe contamination comes from your water? did you check your water as well?
i also face problems with my DNA ladder. the larger bands of the DNA ladder are unable to resolve properly, resulting in a smear. the gel run was performed at 100V, 400mA for approximately 70 mins in a 1.5% agarose gel (TBE buffer)
any suggestions ?
1) Change Taq polymerase. It's a real crap when it happens, but it happens.
2) Pipette mix in reverse direction - first negative sample, then the others.
3) Change pipettes. Are you using the same pipettes for pipetting PCR product and making your mix? They might be contaminated as well.
You can decontaminate it with DNA away or similar product.
The ladder - it looks for me it's either degraded, or too much on the gel.
Hi!
I had a similar problem, but with some differences.
I posted for help in the 'molecular biology' section.
Lately, I had several bad results with electrophoresis run.
I always used 1.5% agarose gels, 80 V and added Sybr Green directly to ladders and samples before loading. I can't use Ethidium because of safety issues.
I began to find something like a big bright band at the end of the run of both ladders and samples, as you can see in the picture. In that case, you can see the ladder, 8 negative samples, the negative control (water) and the positive control (500 bp band).
I thought it could depend on the staining procedure, so I began to perform post-run staining with Sybr Gold. I've tryed from 10' to 30'-long stainings of the gels without improvements.
It can' depend on the loading dye (orange/blue) because it's added to ladder but not to the samples (I use a PCR master mix already containing dyes), and it can't be primer dimers, because in that case it would not be visible in the ladder run. In addition, I found this fake band in several PCRs, so it's not specific of this particular reaction or samples.
It seems to be less intense in the negative control (water, penultimate lane) and in the positive samples.
What do you think it is? What can I do to avoid it?
Thanks for any suggestions!
Can you please tell me your opinion?
Thank you!
Ila