pipetter - (Mar/12/2008 )
My boss don't believe there is such thing of pipetter contamination, and our previous guy seemed did not use them carefully.
We consistently get PCR product in no template control (water instead of DNA template) and got unexpect band on genotyping.
I think it is pipetter contamination since that is the only thing we have not changed.
Is there anyway to prove that?
Where I can gel protocol to clean up pipetter by ourselves?
Basically we have changed basically everything from water, PCR reagent, to primer.
If our pipetter is OK, what went wrong that cgive us the PCR product in no template control reaction.
How to do test to find out what is the problem.
Because next thing going to change on my boss's list is ALL PEOPLE in the lab.
The instructions that come with your micropipetter should include directions on how to clean your pipette, as well as what parts, if any, are autoclavable. The pipettes we use are able to be cleaned with 70% ethanol, and the non-electric pipettes can all go into the autoclave, though they have to be recalibrated afterwards.
Since you're getting a positive result in your negative control, your group members' aseptic technique should also be reviewed to see if that's the source of contamination.
A quick way to show if the pipette is the problem is to use someone else's pipettes, in a different location, and in your PCR space. That way you'll see if the room is the fault, or if it's the pipette.
There are many things you can do.
1.) Use somebody else's pipettors and set PCR (keep everything same except pipettors and gloves)
2.) What is the size of the DNA band you are getting in negative control? There might be the chances of DNA contamination of your enzyme.
If you think it is the pipettor getting contaminated from another user, you can always try using aerosol resistant tips.
you could also use something like DNAzap to clean your pippetes, i think that one is from ambion, there are other brands also to get rid of DNA or RNA contamination. it's easy to use, just wipe the pippetes with a clean cloth with the solution.
Ya...I agreed. Aerosol resistant tips can minimise the possibility of pipette contamination.
Have you tried doing this in a pcr hood? Be sure to clean it prior to using but that should help.