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PCR negative control contamination


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#1 Dave_Kub_11

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Posted 13 February 2010 - 01:11 PM

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!!!

#2 Superman

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Posted 14 February 2010 - 03:05 AM

Hiya,

New primers is a good idea; it may well be that. You should UV all of your reagents and equipment; water, tips, pipettes etc. I would buy a new bottle of DNA and RNA free water too and aliquot it out under a hood.

Are you using filter tips; if not try it with those? Also prepare your mastermix and plate it out under a hood. Also be careful when dispensing your tips that it is not near the tip box.

Finally, try doing your negative controls first and do 2 to be sure.

If that does not work then try changing/reducing the number of your cycles for PCR.

Thats all I can think of.

Cheers,
Superman

#3 Dave_Kub_11

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Posted 14 February 2010 - 03:31 AM

Hiya,

New primers is a good idea; it may well be that. You should UV all of your reagents and equipment; water, tips, pipettes etc. I would buy a new bottle of DNA and RNA free water too and aliquot it out under a hood.

Are you using filter tips; if not try it with those? Also prepare your mastermix and plate it out under a hood. Also be careful when dispensing your tips that it is not near the tip box.

Finally, try doing your negative controls first and do 2 to be sure.

If that does not work then try changing/reducing the number of your cycles for PCR.

Thats all I can think of.

Cheers,
Superman



Hi Superman,

Thanks for the advice,

I have attached the gel in a powerpoint file to this post for someone to have a look at.

After noticing contamination in the first round of PCR I figured it might've been my autoclaved water, so I switched to nuclease-free. Still got the band popping up! I ran 4 negative controls!!! all gave a signal :-(

I have a wealth of experience in genotyping several mouse lines in the lab I used to work in and never worked under a hood during the setting up of the reaction. So I don't know if the problem is there. I've never worked with lacZ before prior to now.

The number of cycles for the extension is quite low (25) so I wonder if increasing them might eliminate this non-specific band?

Thanks,

Dave

Attached Files



#4 Superman

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Posted 14 February 2010 - 08:38 AM

Hiya,

New primers is a good idea; it may well be that. You should UV all of your reagents and equipment; water, tips, pipettes etc. I would buy a new bottle of DNA and RNA free water too and aliquot it out under a hood.

Are you using filter tips; if not try it with those? Also prepare your mastermix and plate it out under a hood. Also be careful when dispensing your tips that it is not near the tip box.

Finally, try doing your negative controls first and do 2 to be sure.

If that does not work then try changing/reducing the number of your cycles for PCR.

Thats all I can think of.

Cheers,
Superman



Hi Superman,

Thanks for the advice,

I have attached the gel in a powerpoint file to this post for someone to have a look at.

After noticing contamination in the first round of PCR I figured it might've been my autoclaved water, so I switched to nuclease-free. Still got the band popping up! I ran 4 negative controls!!! all gave a signal :-(

I have a wealth of experience in genotyping several mouse lines in the lab I used to work in and never worked under a hood during the setting up of the reaction. So I don't know if the problem is there. I've never worked with lacZ before prior to now.

The number of cycles for the extension is quite low (25) so I wonder if increasing them might eliminate this non-specific band?

Thanks,

Dave


Cool. Just make sure you use UV'd nuclease free water when you make up your primers.

Yea; try increasing the number of cycles to 35-40.

#5 Dave_Kub_11

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Posted 15 February 2010 - 10:19 AM

Repeated the PCR with new primers, new bottle of nuclease-free water, new gloves etc. and still got the band appearing in my water control. I'm going to lose it if I can't get this sorted!!!!!!!!!!!!!!!!!!!!!

#6 Superman

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Posted 15 February 2010 - 02:43 PM

Try 35-40 cycles.

#7 Dave_Kub_11

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Posted 18 February 2010 - 12:14 AM

I dropped the DMSO from the PCR mix and used different pipettes that were cleaned. STILL have contamination. I am beginning to think its a problem with the program as I have tried everything fresh.

My boss seems to think increasing the extension to 35-40 cycles will just amplify up the contamination further. I currently have it at 25 cycles. The annealing stage goes down by 0.5 degrees per cycle for 12 cycles.

#8 laurequillo

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Posted 18 February 2010 - 01:23 AM

I dropped the DMSO from the PCR mix and used different pipettes that were cleaned. STILL have contamination. I am beginning to think its a problem with the program as I have tried everything fresh.

My boss seems to think increasing the extension to 35-40 cycles will just amplify up the contamination further. I currently have it at 25 cycles. The annealing stage goes down by 0.5 degrees per cycle for 12 cycles.


Once I had the same problem, and I bought a solution called DNAator (or something like that....) to remove any DNA from my surfaces and from the pipettes...So, I just cleaned everything with that solution (everything!), I used the UV, new tips with filters, new water and then I ordered new reagents and primers (because if the problem was in your pipettes maybe you contaminated the new reagents already). And at the end it dissapeared...(it was just 1 month of work....)
"He must be very ignorant for he answers every question he is asked" Voltaire

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#9 Dave_Kub_11

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Posted 20 February 2010 - 03:06 AM

I think I managed to solve the problem. I altered the program slightly and managed to get rid of the band in my negative control yet still amplify the transgene in my positive at the required base pair size. I ran 3 positive controls just to make sure and they all came up. However, my biopsies I ran all appear to be negative for the transgene as their was no band (they definitely ran as there are primer dimers) so I guess I could take that result as gospel that they are negative. Worst comes to worst, I just stain them for lacZ.




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