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PCR: always got band in the negative control


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#1 Ah-Do

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Posted 05 May 2011 - 04:21 PM

I cloned a construct containing the gene of interest. We generated a transgenice mouse line from this construct. The genotyping is routinely done by our lab technician and the results are all fine.

I am now trying to establish a cell line from this construct. But Iíve been getting problem in PCR for a long time.

I extracted DNA from the transfected and untransfected cells using Qiagen DNeasy Blood & Tissue kit and Extag to run PCR. I use the original construct as the positive control, water, DNA extracted from untransfected cells as the negative controls. However, I keep getting bands in both negative controls. I have changed the dNTP, buffer, water, primerÖetc. everything I can think of that might be contaminated. I also extracted DNA again from freshly prepared cells. But, Iíve been trying for weeks, and the results are always wrong.

So, I gave the DNAs (from transfected and untransfected cells) to our technician. She ran the PCR for me using her own primers and PCR kit that she uses for the mouse genotyping.
She used the DNA extracted from the tail of the transgenic mouse as the positive control, DNA from the wildtype mouse as the negative control. But, she also got band in the DNA from untransfected cell.

Iíve been trying to solve this problem for a long time. But I still cannot think of any reason. Can anyone help me figure out what is wrong? Thanks a million.
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#2 bob1

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Posted 05 May 2011 - 04:39 PM

Try cleaning your pipettes, especially if you are using them when you are extracting the plasmid from the bacteria - it is quite possible that you have contaminated them during this proceedure.

YOu could also try using pipettes from another lab, and/or setting up your PCR in other labs.

Have you replaced your tubes? - lots of people forget this one.

#3 phage434

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Posted 05 May 2011 - 05:17 PM

You may want to run the PCR on a qPCR machine, which would differentiate large amounts of template from minor contaminants.

#4 Ah-Do

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Posted 06 May 2011 - 04:16 AM

Try cleaning your pipettes, especially if you are using them when you are extracting the plasmid from the bacteria - it is quite possible that you have contaminated them during this proceedure.

YOu could also try using pipettes from another lab, and/or setting up your PCR in other labs.

Have you replaced your tubes? - lots of people forget this one.


Hi, thank you for your reply.

I did clean the pipettes, and i also used filter tips. The DNA extraction and the plasmid DNA are not extracted at the same time. the plasmid DNA was frozen in -20C for few weeks before the PCR.

The lab technician is doing the genotyping everyday but she doesnīt have the problem on her samples. But once she ran my samples, the problem occurs.

What do you mean by replacing the tubes? When should i replace the tube, and with what?

I also asked my colleague to set up the PCR and run PCR for me, but still got the same problem - bands in both negative controls..........

#5 BioMiha

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Posted 06 May 2011 - 04:57 AM

We had the same problem and while it frustrated me deeply I could never figure out why this happens. We changed/cleaned everything, even performed the negative control PCR in a different institution, but we always got the same bands. I (and I also got the same comments from other people who have had the same problem) am of the opinion that if you reamplify the same amplicon many times you run a very high risk of contaminating your samples. PCR is very sensitive and can amplify very small amounts of DNA efficiently (God knows where they come from), so your best bet is to either amplify a different region. I know it sucks but in our experience this menace cannot be stopped.
Good luck to you.
Miha

#6 Ah-Do

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Posted 06 May 2011 - 03:21 PM

We had the same problem and while it frustrated me deeply I could never figure out why this happens. We changed/cleaned everything, even performed the negative control PCR in a different institution, but we always got the same bands. I (and I also got the same comments from other people who have had the same problem) am of the opinion that if you reamplify the same amplicon many times you run a very high risk of contaminating your samples. PCR is very sensitive and can amplify very small amounts of DNA efficiently (God knows where they come from), so your best bet is to either amplify a different region. I know it sucks but in our experience this menace cannot be stopped.
Good luck to you.
Miha



Hi, thank you for your suggestions!

but, what do u mean by "if you reamplify the same amplicon many times you run a very high risk of contaminating your samples"? is it the problem of primers?

Our lab technician actually used different primer pairs than mine. The primers that i used should give me a ~600bp band. The primers that technician used should give a ~300bp band. However, i got a 600bp band in the negative controls, and she got a 300 bp band too.......

anyway, i think i will design new primers and polymerase and keep trying...

#7 bob1

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Posted 06 May 2011 - 05:15 PM

By tubes I meant the tubes you are using for PCR and/or extractions.

BioMiha means that the 600 bp you are amplifying (as you have amplified it a lot before) is capable of being spread around the lab thereby contaminating lots of stuff, so changing one thing at a time may not lead to a resolution of the contamination. Remember in theory you only need one copy of the amplicon (result of the PCR) to be able to amplify the DNA.

Your best bet is to get in all new primer stocks and other reagents. Get some clean water, from another lab preferably, clean all your pipettes again, clean your bench, get new tubes, lab coat, gloves, and move into a new lab. Do NOT take any DNA into the new lab. In the new lab, make new primer stocks and mix your PCR reagents. Go back to your old lab and add DNA. Run the PCR, and run the gel.

Note that you might need new samples of DNA, which you may need to extract in a different lab again to prevent contamination of these samples.

#8 Ah-Do

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Posted 07 May 2011 - 07:51 AM

By tubes I meant the tubes you are using for PCR and/or extractions.

BioMiha means that the 600 bp you are amplifying (as you have amplified it a lot before) is capable of being spread around the lab thereby contaminating lots of stuff, so changing one thing at a time may not lead to a resolution of the contamination. Remember in theory you only need one copy of the amplicon (result of the PCR) to be able to amplify the DNA.

Your best bet is to get in all new primer stocks and other reagents. Get some clean water, from another lab preferably, clean all your pipettes again, clean your bench, get new tubes, lab coat, gloves, and move into a new lab. Do NOT take any DNA into the new lab. In the new lab, make new primer stocks and mix your PCR reagents. Go back to your old lab and add DNA. Run the PCR, and run the gel.

Note that you might need new samples of DNA, which you may need to extract in a different lab again to prevent contamination of these samples.



Hi, thank you for your suggestion. I will order new primers and all stuff. hope the problem will be solved.

one last question, how do i clean the bench, pipette etc.? is 70% EtOH enough for cleaning? or should i use any special solution/buffer?

#9 bob1

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Posted 07 May 2011 - 03:32 PM

70% won't get rid of DNA, just precipitate it. You can use 0.1 M HCl to acid hydrolyse the DNA. Remember to wash off the HCl before using the bench. Make sure you decontaminate your bench regularly, I would recommend doing this at the end of each working day. The DNA will also be in the air and all over the lab - so wiping down only your bench may not do much.




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