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PCR contamination


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19 replies to this topic

#1 tantao

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Posted 29 August 2010 - 07:07 PM

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

#2 Kaioshin

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Posted 29 August 2010 - 07:28 PM

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.

#3 tantao

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Posted 29 August 2010 - 08:12 PM


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.

#4 Ameya P

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Posted 29 August 2010 - 09:49 PM

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

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#5 criscastells

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Posted 30 August 2010 - 01:27 AM

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

#6 tantao

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Posted 30 August 2010 - 02:06 AM


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.

#7 gebirgsziege

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Posted 30 August 2010 - 02:43 AM

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.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#8 tantao

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Posted 30 August 2010 - 05:01 AM

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!!!

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Edited by tantao, 30 August 2010 - 05:03 AM.


#9 ranvi

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Posted 30 August 2010 - 05:33 AM

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

#10 tantao

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Posted 30 August 2010 - 05:37 AM

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.

#11 gebirgsziege

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Posted 30 August 2010 - 05:40 AM

sorry so my post was then not much help for your problem, sorry for this.

It looks as if you got a contamination in your chemicals, have you tried to change them and are you sure that nobody else used your chemicals?
And second: autoclaving does not necessary destroy DNA contamination on you pipetts etc., you should try and clean them with bleach, DNA-off or something similar. Do you have some of purchased ultraclean certified DNA/RNA free water in your lab (often also comes as component of extraction purification kits)? Try to use this water for the negative control, if possible try to do your PCR with chemicals from another lab that does not work with mice using their equipment....

Last but most important:can you post your PCR protocol? If the annealing temp is too low you might get unspecific priming with some DNA contaminiation. If possible run a gradient PCR with water only to see at which temp the band is disappearing. If the times are too long there might also be a problem with specificity.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#12 Kaioshin

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Posted 30 August 2010 - 05:42 AM

Your primer stock could be contaminated. For PCR primers, I usually keep a concentrated stock and a more dilute working stock. Do you have a more concentrated stock that you can make a new working stock from? This would be a quick test

#13 tantao

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Posted 30 August 2010 - 05:52 AM

sorry so my post was then not much help for your problem, sorry for this.

It looks as if you got a contamination in your chemicals, have you tried to change them and are you sure that nobody else used your chemicals?
And second: autoclaving does not necessary destroy DNA contamination on you pipetts etc., you should try and clean them with bleach, DNA-off or something similar. Do you have some of purchased ultraclean certified DNA/RNA free water in your lab (often also comes as component of extraction purification kits)? Try to use this water for the negative control, if possible try to do your PCR with chemicals from another lab that does not work with mice using their equipment....

Last but most important:can you post your PCR protocol? If the annealing temp is too low you might get unspecific priming with some DNA contaminiation. If possible run a gradient PCR with water only to see at which temp the band is disappearing. If the times are too long there might also be a problem with specificity.

gebirgsziege,Thank you very much! Only me use these buffers. and I also use UV to bleach the pipetts for 30min
my PCR protocol as follow
1.95℃ 2min
2.95℃ 30s
3.55℃ 30s
4.72℃ 30s
repeat step 2-4 35 times
5.72℃ 10min

#14 tantao

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Posted 30 August 2010 - 05:54 AM

Your primer stock could be contaminated. For PCR primers, I usually keep a concentrated stock and a more dilute working stock. Do you have a more concentrated stock that you can make a new working stock from? This would be a quick test

Dear Kaioshin,
I order the new primer and use the new one to run the PCR. but the ghost band is still there.

#15 gebirgsziege

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Posted 30 August 2010 - 05:57 AM

55°C is not too high, although not too low either. If possible try a gradinent PCR, or if you cannot, try 60 °C with a real sample and the contaminated water....

edit: try to clean the pipett in the inside as well!

Edited by gebirgsziege, 30 August 2010 - 05:58 AM.

A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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