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-   -   PCR contamination (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/3493-pcr-contamination.html)

Doohe* 06-29-2008 09:31 AM

PCR contamination
 
I have a problem during PCR program. add templete DNA, dNTP, primer,

polymerase, etc..to ep tube. then I forgot to add oiling U-U:mellow:

so.. I am nervous for fear of contamination.

Does it matter of this situation? If you shoud be clear up this problem, I will be rearlly preciative of your comment.^_^

admin 07-02-2008 01:06 PM

Re: PCR contamination
 
Hello Doohe,

welcome to the forum.

Its ok Doohe I am sure you should be ok. Always try things thats how everyone learns. Let us know how the PCR goes and post a gel picture and we can take a look and help.

When you say you forgot to add oiling U-U do you mean you forgot to add oil to the PCR machine?

:)

keithbrent2000 08-05-2008 04:38 PM

Re: PCR contamination
 
There shouldn't be a problem with contamination. I think that the oil is more to prevent the liquid in the PCR tube from evaporating when the solution is being heated. If your PCR machine can set up a heated lid while running, that might also prevent evaporation.

saly 08-29-2008 12:24 AM

Re: PCR contamination
 
Please i need the help in my problem:
i design primer but when i made PCR, i found may be formation to
primer dimer under the louding dye but the band of primer dimer under
the negative control ( without RNA ) was small in size than the band
under the sample ( more thick and may be 2 band more closed ). i
belived fond 2 band in sample because the negative control and sample
contain same band in the below but found band upper it in the sample
( may be more thickness )at the same conditions. i tried at 47, 52,
55oC annealing. all tempreature give same result with diffrence in 47
the band under negative control more faint but at 55 the band under
sample more thick and light.
please, i need to know this difference meanse any thing or the
difference in size between the band under control and sample don't
means any thing and the 2 bands are primer dimer only.
Thanks for you

sg30 08-29-2008 12:17 PM

Re: PCR contamination
 
Hi,
I have been getting bands in my control (everything except template DNA)..and when that happens, all the other actual samples also give the exact same bands...and it's an all or none pattern that I have been observing...either all the samples show that sharp bright band that the control has,or at other times all of them show up the correct profile and the control is clean...
i have aliquots of all the ingredients that don't last for more than one PCR run so there is no one 'batch' that i can trace the contamination back to...is it possible that the diluted primers are degrading real fast because one pattern i have noticed is that the first run with a particular primer is alwasy good with all the samples and subsequent runs get bad ..but will degraded primers give bands in the control??

Inputs please!

Thanks,
sg30


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