problem with a negative controls
nospam at spam.dk
Sat Jul 31 06:43:07 EST 2004
.fm is micronesia
"Wolfgang Schechinger" <hubahopp at gmx.de> skrev i en meddelelse
news:22.214.171.124.20040731022008.00eee438 at pop.gmx.net...
> Dear Anna,
> as you probably have a very sensitive protocol, I assume, it's some carry
> over phenomenon or a contamination in any reagent. You could try to reduce
> sensitivity (at least for troubleshooting purposes) by simply reducing the
> number of cycles. How do you determine the treshold GMO / no GMO?
> You might need to clean your pipets, labware (maybe even the disposable
> pipet tips) and bench with 5% hypochlorite (AKA housheold bleach) as UV
> admittedly kills bugs but not really destroys DNA (just crosslinking and
> making T-dimers).
> The best solution for the future might be to prepare your negative
> in a separate room or even better separate building that *NEVER* saw any
> these GMOs you're testing for, using a laminar flow or similar device
> (glovebox) that prevents even atmospheric dust (GMO pollen, if applicable,
> microparticles from sample preparation) to find its way into your tubes.
> course, you'll need second sets of pipets, gloves, lab coats, face masks,
> tips and reagents that never came near your samples to be tested. And
> you'll need to prepare your negative controls before you enter the "hot"
> Reads like Geneticists Paranoia, doesn't it?
> Unfortunately, I know some people who got almost crazy with similar tests
> because of the same reason.
> If you're using supposed non-GMO products as negative controls it could
> simply mean that they contain GMO below the official treshold or they are
> fake or wrongly labelled.
> PS for a *real* +/- confirmation, you'll need a realtime PCR machine to
> measure melting curves, I think (unless you want to analyse (RE / sequence
> / clone) the band from each positive sample. So you might divide your PCR
> in 2 and do some digests with 1 sample.
> Best regards and good luck,
> PPS - where in this galaxy is .fm?
> At 11:40 27.07.2004 -0700, Anna wrote:
> >I'm new worker in molecular labolatory, testing food products for
> >conataining GMO. I use kits for DNA isolation and naxt kits to do PCR
> >reaction. I have two negative (from isolation and before PCR), and one
> >positive control. I receive bands in both of my nagative controls
> >(only in reactions with primers from non-GMO products)!!! I'm using
> >pippetes with filters, wearing gloves, and also using UV lamps. I'm
> >always doing my negative controls first, then all probes and as the
> >last one positive control. Is that possible that I do something wrong
> >or my reactions parameters are bad? Is that possible that this is
> >primers internal reactions?
> unlikely if the bands have the correct size. Only way for confirmation is
> cloning and sequencing
> What can I do to get my negative controls
> Add DNAse... NO NO!!! Don't do it, of course!
> >Please help, and give me some advices!=20
> >Thank you !!!
> Wolfgang Schechinger
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