Persistant p53 Genotyping PCR Contamination

jm317 at cam.ac.uk jm317 at cam.ac.uk
Fri May 2 05:29:30 EST 2003


I replied early but the post wasn't posted ?

Anyway, Thanks for you help in this problem.  I've previously tried
titrating UV dose and ended up with either no product or just faint
contamination.  Water is MQ straight from purifier.  As to mispriming PCR in
negative control ? but there is no template in negative control  unless
oligos can make a product exactly same size ?   Tried cleans Gilsons, using
filter tips as well.   Yesterday, I used fresh promega  mastermix PCR and
fresh aliquot of primers both previously never opened and used the water
that came with the "kit" still had faint band - PCR tube straight from fresh
unopened bag of PCR tubes. Only contaminating thing is lab or me as far as I
can see , used different PCR machine.

Assuming it is PCR contamination I have redesigned the FWD primer 20bp
upstream of the target sequence to prevent it from amplifying any existing
PCR derived contamination  and will let you know. I will also try another
lab.  I'm also having my primers reconstituted at source and aliquoted for
me.

As UNG doesn't help with products already generated using Thymidine, would
it be practical to add Thymidine-DNA glycosylase to the PCR tube prior to
PCR to ensure no template was in the tube and PCR using dUTP instead as the
product is only being used to identify presence or absence of the product ?

Thanks


John
----- Original Message -----
From: "Duncan Clark" <junk@[127.0.0.1]>
Newsgroups: bionet.molbio.methds-reagnts
Sent: Thursday, April 24, 2003 10:43 AM
Subject: Re: Persistant p53 Genotyping PCR Contamination


> Historians believe that in newspost
> <vuywuhldnmu.fsf at suspiria.ai.mit.edu> on Wed, 23 Apr 2003, Tom Knight
> <tk at suspiria.ai.mit.edu> penned the following literary masterpiece:
> > If you
> >really need sterility, order the gamma irradiated tubes and tips.
>
> But gamma irradiation doesn't trash DNA. We get certain plasticware we
> buy in gamma irradiated by taking it over to Isotron in the UK. It
> really gets blasted and the polypropylene comes back slightly yellow -
> gamma irradiation basically ages the plastic.
>
> Anyway we once added in some lamdba DNA into various tubes and ran a
> real-time before and after irradiation. No real difference for small
> targets of less than 250bp.
>
> It probably introduces ss nicks but that won't stop the PCR.
>
> Duncan
> --
> I love deadlines. I especially like the whooshing noise they make as
> they go flying by.
>
> Duncan Clark
> GeneSys Ltd.

<jm317 at cam.ac.uk> wrote in message
news:b86637$97t$1 at pegasus.csx.cam.ac.uk...
> Hi
>
> I have an ongoing persistent PCR contamination.  We have a colony of p53
> knockanimals and genotype according to the JAX protocol with 2 primer sets
> which are used in separate rather than a multiplex reaction so as to
improve
> resolution.  Homozygous contamination at 280Bp occurs frequently rendering
> accurate genotype impossible.  All the usual preventative measures
> fails...if anyone can come up with something I've missed and has had
similar
> experience I would be grateful for their comments.
>
> 1. Aliquot primers, dNTPs, Taq even under flowhood
> 2. Sterile Filter tips
> 3. Sterile, UV'd Water
> 3. Sterile/autoclaved/pyrogen free PCR tubes
> 4. Sterile /new Gilsons
> 5. Dnased blocks,bench, centrifuge etc
> 6. Clean Lab coats, gloves etc
> 7. Setup is on separate bench
> 8. Diluted DNA template concentration
>
> I just can't understand where the contamination, which I believe to be PCR
> rather than genomic DNA is getting in.  Even reducing the number of cycle
> down to 20 makes no/little difference.   Homo contamination will make
every
> WT a hetrozygous which is not good!
>
>





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