PCR contamination - does autoclaving help? - (Apr/15/2006 )

Hi all

I can now realize that how difficult and troublesome it can be to get rid of PCR contamination, I have read all the postings in this forum and tried most of the things but I couldn’t get rid if those contaminants, the bands I am getting in the contaminants is faint but visible, reducing the cycles will just be denying the fact that there is no contaminant, when there is.

Two times I have thrown all the samples, and autoclaves everything possible but I failed to find the location of contamination, my results are coming like this positive is bright big band negative is faint but visible band. I am loosing enthusiasm of working, I am the only one working in my lab and my all reagents and pipettes are not handled by anyone other then me.

I am working on BCSP31 region of Brucella melitensis for diagnosis of the disease.

Any kind of slightest help will be a great help, I just wannna get out of this at any cost, I already lost my four months in this. Cant loose any more,

Try *not* autoclaving things. Autoclaves are full of, you know, chemicals and things. I'd suggest clean plastic ware, barrier tip pipets, reagent quality water, etc. not go near an autoclave. The purpose of an autoclave is to kill things, not to make things chemically pure. In particular, DNA probably survives in autoclaves in quantities large enough to be PCRed.

I think Phage434 raised a very good point. PCR contamination is not a problem for me even I don't autoclave my stuff. I know that if you try to amplify something repeatedly and intensively contamination is easy to creep in.

Some good practices:

1. Assemble your PCR in a separated area.
2. Use a new pipette tip every time you touch you stock solution. For example, you are doing two sets of PCRs and are preparing two mastermixs, have added water to the first tube, if the tip has touched the tube, you should use a new tip to add water to the 2nd tube. The same is true of adding buffer, dNTPs, primers, Taq, etc. This will help keep your reagents clean.
3. Always add template last.
4. Using aerosol resistant pipette tips may help, but is not a must.

Hope that helps.

Could it be that the false products you're getting are not the result of containination, but result from homologous sequnces in your template? What is your template -- is it genomic DNA? Have you tried a different set of primers, checked your primers against your target, etc.?

hi
if your are using for diagnostic purpose better find some other target apart from what you are unsing now. Please change whole PCR set up includig primer , enzymes dntps and even the rack and change the place whre you are presently storing
shashi

I had this question too... if the autoclave really do some thing or is just a waste of time... so I made some tests.
I did some reactions with known targets.
Than I made a series of dilutions and autoclave all this...
Than I try to amplify this autoclaved samples with the same primer that I amplified first. (like a auto-nested pcr)
The autoclaved samples reduced veeeery much the "amplification capacity" of the amplicons.
I´m planing to do some other tests and published that.
My english is terible, i hope you did undertand me.
Well.. what i´m trying to say is:
Autoclave seems to work for decontamination.

Ask me if you have other questions about this test.

Julio

hi
autoclaves do reduce contamination, that's my experience. use a laminar exclusively for PCR, put on uv light for 30 minutes, autoclave tips and tubes used for pcr and also dispose after one use/ wear gloves while pippetting your reagents.
good luck

Hi
I had this question too... if the autoclave really do some thing or is just a waste of time... so I made some tests.
I did some reactions with known targets.
Than I made a series of dilutions and autoclave all this...
Than I try to amplify this autoclaved samples with the same primer that I amplified first. (like a auto-nested pcr)
The autoclaved samples reduced veeeery much the "amplification capacity" of the amplicons.
I´m planing to do some other tests and published that.
My english is terible, i hope you did undertand me.
Well.. what i´m trying to say is:
Autoclave seems to work for decontamination.

Ask me if you have other questions about this test.

Julio