problem with a negative controls

Wolfgang Schechinger hubahopp at gmx.de
Fri Jul 30 19:27:29 EST 2004


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 controls
in a separate room or even better separate building that *NEVER* saw any of
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. Of
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" lab.

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, 
Wo

PPS - where in this galaxy is .fm?

At 11:40 27.07.2004 -0700, Anna wrote:
>Hello!
>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
>clear?

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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