what do you think of this gel ? - (Oct/30/2008 )
Hi guys,
please, have a look to the attached agarose gel (1%) picture attached and let me know what you think of it.
The samples are PCR products, top lane 55ºC as annealing temperature and bottom lane 58ºC as annealing temperature. The faint bands are at the expected size, ~1.5kb (bacterial 16S).
Final chemicals concentration :
-MgCl2 1.5mM
-dNTPs 0.2 mM each
-reverse and forward primers (both fluorescently labeled) 0.2µM each
-BSA 1 µg/µl
-ExTaq 1 unit for 50µl of PCR mastermix
Cycling regime : 30 cycles of 1' denaturation (94ºC), 1' annealing (55ºC usually), 2' extenstion (72ºC). There's also an initial denaturation (3' at 94ºC) and a final extension (10' at 72ºC).
The important thing is that no DNA template was used in this PCR (usually, I use 1µl of the raw DNA extracts (from soil) which falls in the 0.1-1 µg of DNA for most of my samples).
I've had contamination with other primer sets not related at all to bacterial 16S (they were for plants microsats and I was working with plant DNA).
In between, I have changed chemicals, beached the pipettes and the benches.
The smears look pretty weird don't they ? They stop where I should get a band.
I've done PCR for almost 10 years and I never had that before !!!
Any ideas ??
Thanks !
Any idea ?
have you tried tubes (or plates) from a fresh box?
No but I can do that, I have boxes that were never opened.
thanks !
Hi,
isnt it bacterial contamination from the air? I had something similar when working with ITS primers last summer, there was always quite distinct band in negative control. For example, I tried two different PCRs at the same day - one with ITS (rDNA) primers and another one with chloroplast primers, and cpDNA control was always OK, but in ITS was contamination. It might be due to higher specificity of chloroplast primers (only plant contamination can make bands), whereas primers for rDNA might work in wider spectrum of taxa (possibly contamination from pollen grains, fungi spores?)
Jirka
isnt it bacterial contamination from the air? I had something similar when working with ITS primers last summer, there was always quite distinct band in negative control. For example, I tried two different PCRs at the same day - one with ITS (rDNA) primers and another one with chloroplast primers, and cpDNA control was always OK, but in ITS was contamination. It might be due to higher specificity of chloroplast primers (only plant contamination can make bands), whereas primers for rDNA might work in wider spectrum of taxa (possibly contamination from pollen grains, fungi spores?)
Jirka
Could be...
I did this very same PCR last year and it was working fine. Then I started another project on plant microsats. Everything went fine. All of a sudden, the negative controls turned out positive (for microsats PCR). Now I am back to the initial project (bacterial stuff) and here is the contamination again.
When I prepare the PCR I keep the tubes closed and get to open them only to add the mix then again to add the template.
Could I have RNA contamination ?
thanks !
You really got contaminations in microsats negative controls? It is strange, SSR primers are very specific. You mentioned using new reaction components, what about primer stocks or loading buffer? I dont know, maybe try working in flow-box, or another lab, if you have possibility. My friend told me about her problems with PCR - she had contaminations in her lab, but when he changed the lab, it turned out OK...
isnt it bacterial contamination from the air? I had something similar when working with ITS primers last summer, there was always quite distinct band in negative control. For example, I tried two different PCRs at the same day - one with ITS (rDNA) primers and another one with chloroplast primers, and cpDNA control was always OK, but in ITS was contamination. It might be due to higher specificity of chloroplast primers (only plant contamination can make bands), whereas primers for rDNA might work in wider spectrum of taxa (possibly contamination from pollen grains, fungi spores?)
Jirka
Could be...
I did this very same PCR last year and it was working fine. Then I started another project on plant microsats. Everything went fine. All of a sudden, the negative controls turned out positive (for microsats PCR). Now I am back to the initial project (bacterial stuff) and here is the contamination again.
When I prepare the PCR I keep the tubes closed and get to open them only to add the mix then again to add the template.
Could I have RNA contamination ?
thanks !
yes, I did get contamination in microsats negative controls (the band was at the exact same size as in the samples).
At that time I thought that maybe I got a pipette contaminated by genomic DNA. But even after bleaching, using DNA away, etc... the contamination was still there.
About 16S primer stocks : I have not used these for 1 year and at this time I had no contamination. For the loading buffer I am not sure though.
I should try in an other lab. The previous I was working in had a special PCR hood with UV bulb. I rarely got some contaminant back then.
Do you use filter tips? If it is so, contaminated pipette should not be the case, and the problem might be rather in chemicals...
What about DNA extraction? Somebody use grinding with mortar and pestle in liquid nitrogene, and this can cause contaminations by plant powder (that might be the case of genomic DNA for microsats you mentioned).
yes, I did get contamination in microsats negative controls (the band was at the exact same size as in the samples).
At that time I thought that maybe I got a pipette contaminated by genomic DNA. But even after bleaching, using DNA away, etc... the contamination was still there.
About 16S primer stocks : I have not used these for 1 year and at this time I had no contamination. For the loading buffer I am not sure though.
I should try in an other lab. The previous I was working in had a special PCR hood with UV bulb. I rarely got some contaminant back then.
What about DNA extraction? Somebody use grinding with mortar and pestle in liquid nitrogene, and this can cause contaminations by plant powder (that might be the case of genomic DNA for microsats you mentioned).
No, I don't use filter tips, that's why I bleach pipettes often (it's not that I don't want to but my boss thinks they are too expensive... well doing over and over the same PCR is also expensive I think !).
For DNA extraction I use a beadbeater.
I did another PCR yesterday (see gel2). I tried 2 DNA samples with or without BSA and with or without RNase A (in the tubes containing the samples pre-treated with RNase A, there was like a white layer on top of the mastermix at the end of the PCR).
Neg1 and Mix on the gel are the same sample (mix is the leftover of mastermix). One has a band, not the other. It is making me mad.
Help !!!!!!!