my negative PCR control is still positive ! - (Apr/25/2008 )
Hi guys,
I have been through all the posts dealing with this issue and have followed most of the advices I could find. But my PCR negative controls still turn out positive.
I work on plant microsats (a 250bp fragment). What I have done :
-wipe the benches with soap and DNA OFF
-soak the pipettors shafts and tip ejector in DNA OFF (twice)
-autoclaved new tips (unfiltered), and tubes
-ordered new primers and dNTPS
-autoclaved new water and TE buffer (buffer used to resuspend the primers).
-have set up the PCR and added DNA samples in different rooms
After all this cleaning, I ran my first PCR and the gel. In the PCR, I included two negative controls (in one of them, I added water as samples; the second one was PCR mastermix only). As I said, both are positive.
One month ago, this very same PCR was working great.
I use a touchdown PCR protocol.
I am getting desperate !
I add a file (PCR picture). The negative controls are on the right hand side of the gel (first one : mastermix only; second one : water as sample). The other wells are samples.
Any idea ? Advice ?
thanks !
Valerie
Have you tried a fresh batch of enzyme? Maybe that is contaminated?
I forgot to mention that, sorry.
Yes, I also used a new PCR kit. I also prepared fresh BSA fresh DNA dilutions with the freshly autoclaved water.
I used different set of pipettors also to prepare the mastermix and add DNA.
I have been through all the posts dealing with this issue and have followed most of the advices I could find. But my PCR negative controls still turn out positive.
I work on plant microsats (a 250bp fragment). What I have done :
-wipe the benches with soap and DNA OFF
-soak the pipettors shafts and tip ejector in DNA OFF (twice)
-autoclaved new tips (unfiltered), and tubes
-ordered new primers and dNTPS
-autoclaved new water and TE buffer (buffer used to resuspend the primers).
-have set up the PCR and added DNA samples in different rooms
After all this cleaning, I ran my first PCR and the gel. In the PCR, I included two negative controls (in one of them, I added water as samples; the second one was PCR mastermix only). As I said, both are positive.
One month ago, this very same PCR was working great.
I use a touchdown PCR protocol.
I am getting desperate !
I add a file (PCR picture). The negative controls are on the right hand side of the gel (first one : mastermix only; second one : water as sample). The other wells are samples.
Any idea ? Advice ?
thanks !
Valerie
Just curious --why are you using unfiltered tips? You could be contaminating your negative controls with your pipettor if you're setting up your other reactions first and not using filtered tips.
Just curious --why are you using unfiltered tips? You could be contaminating your negative controls with your pipettor if you're setting up your other reactions first and not using filtered tips.
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I totally agree with you about unfiltered tips. I wanted to order filtered one but my PI thinks they're too expensive
Tell your PI that is more expensive repeat the experiments because of contamination. The cost of buy all new again is higher than the cost of the tips with filter. For PCR and RNA experiments use filter tips. If the enzyme, buffer, dntp's, primers, water and any other reagent, materials like tips, tubes etc. are new. There is 2 possibilities the area is contaminated (use a hood or pcr working station and UV for 30 min gloves , tips, water, racks, tubes) or sorry to tell but there is a possibility of not GLP.
we do not have any UV here :-( But I'll try to find some.
I have been doing PCRs in this lab for 1 yr now and they've always been good. It's been bad for about 1 month now.
Could it be the water system ? I just set up a new pcr with water from another source.
I'll also use a new loading dye tube and a new pipettor to load samples on the gel.
I always work with gloves and change them often.
I have been doing PCRs in this lab for 1 yr now and they've always been good. It's been bad for about 1 month now.
Could it be the water system ? I just set up a new pcr with water from another source.
I'll also use a new loading dye tube and a new pipettor to load samples on the gel.
I always work with gloves and change them often.
Autoclaving does not remove the DNA necessarily. Some people classify autoclaves as source for contamination. Perhaps try other source of water (some companies sell it for those reasons). Also the destiller ( or whatever is the name for it, i.e for producing ddwater can be the contamination source).
Can you work in a hood and what about aerosol-dust contaminations? In what room do you extract your DNA (same as the one used for PCR-setup?), and do you use the same pipettors to prepare your PCR's (if so, without filtertips you'r taking a very high risk). anybody else working on genomic material that also has this microsat?
What's your pipetting order?
Can you do a nega control sample (just mastermix), close the tube and then add mastermix to samples and do another nega control reaction (water added) in this order?
Do you share the reagents with many people???if so, make an aliquote of each just for your use.