Pre-PCR and Post-PCR activities in one room
Posted 19 October 2009 - 03:22 AM
Posted 19 October 2009 - 03:31 AM
It will depend on your research too; there are exp. more prune to contamination than others....so maybe your experiment is not that precious when it comes to contamination don't get me wrong; for example microbial community studies from environmental samples are less susceptible for contamination than eg characterising a single copy gene....
Edited by gebirgsziege, 19 October 2009 - 03:32 AM.
Posted 19 October 2009 - 04:44 AM
Posted 19 October 2009 - 04:59 AM
I think you are asking for trouble before it finds you. Worry about your real problems, not the ones you can imagine.
Lobby for filter tip pipette tips, which will be much more important to you than a different room.
Posted 19 October 2009 - 08:53 AM
Thank you guys for all your replies . I am really stressed out and your words relieve me a lot . It feels nice to have someone who can understant me , listen to me and reply me . Thank you all.
By the way , do I have to use filtered tips when handling with all PCR reagents including Buffers and Magnesium Chloride ? Or it's ok to dedicate such tips just for pipetting Nucleic acids ( PCR products , DNA extract, primers , Restriction Digest ...etc ) . I mean what's the point with pipetting Non-nucleic acid reagents such as PCR buffer and dNTP with filtered tips ? Contamination of the pipette with dNTP or PCR buffer or Taq wouldn't harm as long as all the reagents are not contaminated with any source of DNA or PCR products . It's more economic to dedicate filtered tips for only Nucleic acids and not use them for routine pipetting of everything , right ?
Edited by Bassaml7, 19 October 2009 - 08:54 AM.
Posted 19 October 2009 - 01:15 PM
Posted 19 October 2009 - 02:52 PM
We use Virkion (or something like that) to clean everything up before putting it under the hood. Haven't had any major problems yet. The vikion idea came from a person that ran a molecular diagnostics lab down the hall from us. That lab DOES use various rooms for various PCR steps, because of the sensitive nature of disease diagnostics.
But for us, we just do cloning, colony PCR, some occasional qPCR, and don't have much, if any problem having multiple stages in one room. As others have said, you just have to be clean.
Posted 06 March 2011 - 12:39 PM
Posted 10 March 2011 - 08:58 AM
I'm in a forensic lab and of course we have separate room with pressure in the vestibules and if you go to a post PCR lab, you are not allowed to come back in the pre PCR lab until you change cloth and shower.
Carryover will be in a post PCR for sure, now the question is: will you detect it? Because contamination isn't a black and white thing. You understand that if you vary the number of cycles in your PCR or the size of your amplicons, things will change. So I say that you will have problems if the amount of DNA you want to amplify is in quantities close to the amount of carryover in the room. If you work with ug of DNA, you should be fine.