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PCR contamination - (Aug/29/2010 )

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Dear everyone,
The PCR contamination makes me crazy. My band is 107bp, I use ddH2O as template and get the positive result. I change everything: the PCR buffer ,ddH2O and use the filter tips. I also autoclav the pipette. But when I use ddH2O as template ,the band are still there. I do not know what to do know. Can anyone give me some suggestions?
Yours sincerely
Tao

-tantao-

tantao on Mon Aug 30 03:07:20 2010 said:


Dear everyone,
The PCR contamination makes me crazy. My band is 107bp, I use ddH2O as template and get the positive result. I change everything: the PCR buffer ,ddH2O and use the filter tips. I also autoclav the pipette. But when I use ddH2O as template ,the band are still there. I do not know what to do know. Can anyone give me some suggestions?
Yours sincerely
Tao



For starters, you could try ordering primers that are slightly different than the ones you have. Shifted a couple, 5, or 10 bp from the ones you have now. You could be dealing with some type of primer dimer product rather than a contamination of template.

-Kaioshin-

Kaioshin on Mon Aug 30 03:28:21 2010 said:


tantao on Mon Aug 30 03:07:20 2010 said:


Dear everyone,
The PCR contamination makes me crazy. My band is 107bp, I use ddH2O as template and get the positive result. I change everything: the PCR buffer ,ddH2O and use the filter tips. I also autoclav the pipette. But when I use ddH2O as template ,the band are still there. I do not know what to do know. Can anyone give me some suggestions?
Yours sincerely
Tao



For starters, you could try ordering primers that are slightly different than the ones you have. Shifted a couple, 5, or 10 bp from the ones you have now. You could be dealing with some type of primer dimer product rather than a contamination of template.


Thank you Kaioshin. the primer dimer is below 100bp. I do not know what is the problem.

-tantao-

I saw someone mentioning gloves which resulted in contamination in this forum. Use separate pair for gloves to prepare your mastermix and to aliquot it into tubes. Also, get a brand new set of primers (it might be contaminated).

Ameya

-gt_ameya-

gt_ameya on Mon Aug 30 05:49:57 2010 said:


I saw someone mentioning gloves which resulted in contamination in this forum. Use separate pair for gloves to prepare your mastermix and to aliquot it into tubes. Also, get a brand new set of primers (it might be contaminated).

Ameya


If you prepare your master mix in a flow cabinet, make sure that it is completely clean and maintain the filter off.

CRIS

-criscastells-

criscastells on Mon Aug 30 09:27:46 2010 said:


gt_ameya on Mon Aug 30 05:49:57 2010 said:


I saw someone mentioning gloves which resulted in contamination in this forum. Use separate pair for gloves to prepare your mastermix and to aliquot it into tubes. Also, get a brand new set of primers (it might be contaminated).

Ameya


If you prepare your master mix in a flow cabinet, make sure that it is completely clean and maintain the filter off.

CRIS

Thank you Ameya and CRIS. I always make the flow cabinet open. Maybe this the problem. I run the PCR again. Hope everything is ok this time.

-tantao-

when trying to amplify DNA from Bacteria, contamination from Taq production (=DNA from ecoli) in the enzyme preparation can be the problem.
We once had exactly this problem, but after a lot of asking and trying using either Phusion (Finnzymes) or TrueStart (Fermentas) instead of the routinely used cheap enzyme solved the problem.

-gebirgsziege-

gebirgsziege on Mon Aug 30 10:43:59 2010 said:


when trying to amplify DNA from Bacteria, contamination from Taq production (=DNA from ecoli) in the enzyme preparation can be the problem.
We once had exactly this problem, but after a lot of asking and trying using either Phusion (Finnzymes) or TrueStart (Fermentas) instead of the routinely used cheap enzyme solved the problem.

Dear gebirgsziege and everyone,
I repeat the PCR again. I amplify the beta actin of mouse. I use the ddH2O as template. But I also get a band around 100bp which is the correct band. I am really crazy. What is wrong!!!
Attached Image

-tantao-

proceed with your experiment sometimes it will work, when I got the same problem i went forward it was fine

-ranvi-

ranvi on Mon Aug 30 13:33:03 2010 said:


proceed with your experiment sometimes it will work, when I got the same problem i went forward it was fine

Dear ranvi,
I used this primer for real-time pcr. There is not dimer and false positive before. But now everything comes out.

-tantao-
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