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Amplification in water control but not in samples


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#1 frna11

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Posted 14 August 2011 - 08:44 PM

I have some stable cell lines running and wanted to verify presence of Neo insert through PCR. I used standard DNA precipitation technique and ran a PCR with conditions that have worked previously. My water control had the master mix and an extra 1ul of H20 from the same source as used in the master mix. I looked at 7 clones, a negative control (untransfected cell line) and a positive control. When I ran it on a gel I found a band for my water control, my positive control and onen of the 7 clones. I would have thought that if it amplifies from the water control it would amplify from all of the samples? Is that right? Any suggestions for what might have happened and what to do from here would be great. I'm thinking of repeating it..

#2 pito

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Posted 15 August 2011 - 07:05 AM

I dont understand what you mean by "water control" , as far as I understand it the water control is an extra negative control: it does not have DNA in it since you only add mastermix and water in it.

So you should see no results at all in that water control sample.

Are you sure you are not mixing up something here?

And its possible that not all 7 samples are transfected like they should.

Altough: if it worked before.. and now it doesnt.. something is wrong (but you could have figured that out allready if you see a negative control that forms a band)

Edited by pito, 15 August 2011 - 07:06 AM.

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#3 merlav

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Posted 15 August 2011 - 08:38 AM

Usually if the water is contaminated you will see a band in each sample including the NTC (no template control). It looks to me that was a problem with technique (no the pcr, the way you are doing the reaction). In some point you introduce a contamination. Obviosly I can't see you working so I'll make the recommendations base on common mistakes that I usually see that the students do.

1. alicuote and prepare your own working materials because when many peoples use the same reagent there is a chance that somebody contaminates it and then the rest of user will have trouble with their reactions. I always prepare a box for ea. student.
2. prepare the area that you are using for the pcr (decontaminate and place all materials that you will need) so you avoid to stand up to looking for something in the middle of work, thats a way of introducing contaminants to the area and in the reaction. Also is good idea to have a pipet set for pcr use only and when budget allows it use filter tips.
3.Do a master mix of the reaction (all except template), adding a reagent to each tubes rise the opportunity for a mistake and contamination. I always prepare an excel form with the reagent, quantity in each tube and how much I need for Xsamples (plus 1).
4. When placing the tubes on the rack make sure no one falls (if falls throw it away the tubes cost a few cents reactions that fails cost more money).
5. Pipet the master mix to all tubes (if you do it with care you will need 1 or 2 tips only), then add the template (one tip per sample), close the tubes after adding the template.
Hope this help
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#4 frna11

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Posted 15 August 2011 - 11:18 PM

Thanks for the replies guys. And yeah, I was confused at seeing the band in water control but not in the others.. I figured if the water/mastermix is contaminated then everything should have the band.

Just to clarify, I meant that these PCR conditions and primers had worked previously, not that my specific samples had been shown to have Neo before, so it is entirely possible that only one of them has it (although somewhat unlikely as it was selected for).

Our lab has a seperate area for PCR with filter tips and I did make a master mix for it, but I realised that I had used MQ H20 from my bench as opposed to the PCR bench MQ H20, it's possible that that is where the contamination came from, although it doesn't explain it only being in only the water control.. I repeated the PCR and obtained a band for a single sample (the same one) and nothing in the water. Seems like it was just a matter of some contamination there and that my selection did not work.




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