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Help -Water Blank keeps coming up with mutiple bands


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8 replies to this topic

#1 Kate2011

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Posted 29 June 2011 - 04:50 AM

Hi there,

I've been running SRAPs, BOX Elements and MSP-PCR but my water blank keeps coming up with multiple bands.

I've tried (1) new primers (2) new TE to reconstitute the primers (3) new nuclease free water (just opened) (4) different 10 X PCR buffers (5) different pipets and (6) different vials of dNTP.

Additionally I've tried running the PCR at different temperatures (gradient).

Interestingly, others doing PCR in the lab do not have a problem with their NTC coming up positive.

Any suggestions to rectify the problem.

Thanks much

Kate

#2 bob1

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Posted 02 July 2011 - 02:29 PM

It is possible that you have managed to contaminate more than one of your reagents. I would throw out the water, the buffers, the dNTPs, any tubes you are using currently, and the primers (and if you want to, the polymerase too). Now, go to a different lab if you can, and using fresh tips (filtered if possible) and different pipettes set up your PCRs again, testing old polymerase against new.

Note - for PCR most tubes come DNA, RNA and nuclease free, these do not need to be autoclaved before use - this only adds contaminants.

#3 pito

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Posted 02 July 2011 - 03:12 PM

Interestingly, others doing PCR in the lab do not have a problem with their NTC coming up positive.


ask one of them to try it with YOUR reagents.. see what that gives.


Just to rule out any mistakes you make and be sure the reagents are not contaminated.


This doesnt take a lot of time: when they prepare a PCR, let them just do a few with your reagent too.. Its easy, fast.


If they get positive results too.. do as bob1 says.
This would take more time and money of course, but if its needed,its needed.

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#4 Ameya P

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Posted 02 July 2011 - 10:56 PM

Kate,

If you have changed everything and still get bands in your negative control, the problem is probably your technique. I could be completely wrong here, so ignore this post, if I am.

Please check for instances, while you are setting up the PCR, where you could be contaminating your negative control reaction like using the same tip for adding template/ contamination with gloves.

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#5 Kate2011

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Posted 05 July 2011 - 07:34 AM

Thanks for all your replies.

I've already covered all of your suggestions. Still have banding pattern in my NTC. Changed all components, changed individual components. Since we currently share reagents in my lab - Others using them come up with clean NTCs.

Tips are filtered tips. PCR assembled in the biosafety cabinet or at the bench come up with banding in the NTCs.

I'm stumped!

Thanks again for taking the time to help out.

#6 Adrian K

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Posted 05 July 2011 - 11:57 AM

I would like to highlight 2 quotes (from phage434, and Trof) from past posting:


Human cells and DNA are everywhere. How many cycles are you doing? With enough cycles, you could probably amplify a human gene from almost anything. Think about everything that comes close to the tube. Do you autoclave tips or tubes? Perhaps the autoclave is contaminating things. UV treatment of water won't solve contamination problems. Set up and store your reagents away from the cycler, and especially away from any amplified DNA products. Ideal would be a different lab or different bench. Are you certain you have a product, and not primer-dimers?



BioMiha: Was your amplicon by a chance a bacterial gene? Some Taq enzymes produced in bacteria can have contamination by traces of host strains DNA.



Can you possible clone the fragment from NTC, and sequence and blast it and try to figure out what template is that? Perhaps by then you can possible find out where the contamination possibly come from.
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

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#7 Kate2011

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Posted 05 July 2011 - 12:02 PM

Thanks,

You've made two excellent points. Since the MSP-PCR primers are simply small repetitive sequences like (CAG)5 it would amplify almost anything. I did not think about the Tag which could be the issue.

Thanks again.

Cheers

#8 Adrian K

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Posted 06 July 2011 - 08:03 AM

Do update us if you managed to find the culprit. ^_^
Good luck.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#9 Kate2011

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Posted 08 December 2011 - 10:05 AM

After much investigation, I threw up my hands and started from scratch will all new primers, water, taq etc... There was no bands in the water.

Hurray!!!




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