my negative PCR control is still positive ! - (Apr/25/2008 )
In the last PCR, there was just water and PCR reagents and I have used different waters and different autoclaves (I have tried to use most of the combinations water x autoclave). Most often the contamination is there when I use the water we have in the lab. I have not run all the combinations yet on the sequencer but will do as soon as it is available.
Any suggestion ?
Thanks !
Valerie
This sounds as if the autoclave is at least one of the contamination source. For work I would at least use different lab desks for every part of the work (dna extration, pcr setup, etc.) and clean everything carefully before work perhaps also with a DNA-killer chemical (some companies sell, e.g. DNA-Ex), and clean the pipettes before use (EtOH and the DNA killer if possible). Buy PCR-grade water. Have tip boxes and all tubes only for PCRs (if autoclaving of them is necessary, then with an uncontaminated autoclave). Perhaps clean the autoclave in a similar way.
I don't know if many researchers try to sequence their negative controls if there is no band. Don't develop a paranoia
.You're right I am becoming paranoid
But the fact is that the sequencer detects peaks in the negative controls and at the same size as in my samples. So it makes my life complicated when I tried to call alleles if all the peaks are there
Do you have any experience with DNa-killer solution ? Iknow DNA away, DNA off and you mentioned DNA ex ? One you would recommend ?
thanks a lot !!!
I used DNA-ExitusPlus from Applichem, but I guess the solutions from other companies are all quite similar, most important they're non-toxic and non-corrosive (careful with pipettes etc).
BTW if you sequence your negative controls, then you should know what it is i.e. if there are microsats in the contaminations or not...
Stupid question: are the plants you are working with flowering at the moment? If you don't use a hood, maybe some pollen flying around in the air are contaminating you negative controls ????
Not joking. We had a heavy pollen load two years ago and for two weeks there seemed to work no PCR at the whole university, because the pollen were interfering with the experiments!
Not joking. We had a heavy pollen load two years ago and for two weeks there seemed to work no PCR at the whole university, because the pollen were interfering with the experiments!
that's good to know !
Well, th time the plants were in the lab was mid-march. I don't recall they were flowering at that time.
I am still struggling with the contamination. I thought I did trace it back to both the water system and the autoclave but, when I try to repeat the test, it comes out totally different.
We just got a UV bulb, that's my last chance.
By the way, I was wondering if there could be something wrong with the thermalcycler itself.
Barrier tips. All new primers, enzymes, buffer, water, tips, tubes, caps. Never let them near an autoclave (they don't need to be sterile). Gloves.
The problem is almost always contaminated reagents, usually because barrier tips were not used in the past.
Not joking. We had a heavy pollen load two years ago and for two weeks there seemed to work no PCR at the whole university, because the pollen were interfering with the experiments!
that's good to know !
Well, th time the plants were in the lab was mid-march. I don't recall they were flowering at that time.
I am still struggling with the contamination. I thought I did trace it back to both the water system and the autoclave but, when I try to repeat the test, it comes out totally different.
We just got a UV bulb, that's my last chance.
By the way, I was wondering if there could be something wrong with the thermalcycler itself.
There was an issue with pcr tubes and contamination etc. Perhaps also clean the well and use tubes that close tight (and be sure that they are really completely closed)
to check if your pcr machine is ok the best thing is to try another one, in a different lab, if they're kind enough to borrow it.
I am planning a new test using UV, bleach (10% probably) and new PCR tubes both on our PCR machine and one from an other lab. Hopefully something good will come out !
I am planning a new test using UV, bleach (10% probably) and new PCR tubes both on our PCR machine and one from an other lab. Hopefully something good will come out !
be careful with bleach! When you dont remove the bleach properly, your negative control will be negative but your positve control too
just another idea - as your PCR-product is quite short: are you sure your primers do not from dimers and you are getting chimeric PCR-products? Have you tried your reagents with another primer/template combination to check for impurities?
g
Again, use filter tips. Tell your PI that buying filter tips is far less expensive than all the wasted reagents from repeated contaminated experiments.
1) Are the areas separated enough , PCR1, PCR2 & PCR3?
2) Prepare 1st. your Master Mix in PCR 1. Let your tubes sit on PCR1 area on cold until ready to use.
3) Change your labcoat and gloves.
4) Go to PCR 2, prep your samples.
5) Discard gloves and change labcoat, bring your MM to the PCR2 area. Use gloves from PCR1 to pick up MM.
6) Add samples to your Master Mix (MM)
7) Take samples to amplification.
It may help?
Floppy