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Pre-PCR and Post-PCR activities in one room


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9 replies to this topic

#1 Bassaml7

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Posted 19 October 2009 - 03:22 AM

My Professor told me I can perform the two sort of activities in a single room . I failed to persuade her that it's much safer to at least seperate pre-PCR from Post-PCR activities . Some say 4 rooms have to be used . One for PCR reaction set-up , a second for DNA extraction , a third for the thermocycler and the fourth for post amplification activities ( electrophoresis ) . I have shown her many reviews and many diagrams for a PCR laboratory layout but still she's persistent that all activities can be done in a single room . In fact we don't have much space in our lab but we do have three separate rooms . The thing is she just intends to use these rooms for other things . In the End it is my research thaty will suffer from such practise . I am sorry to disturb you all with this private story but I am totally down . And I am just looking for someone who can understand what I am talking about , just to vent . By the way , have anyone here tried such a Practise ( performing everything in a single room ) For how long did it work before contamination shows up. At least I just thought it's best to restrict the electrophoresis area since PCR tubes are opened there and PCR products are the major risk . I am so worried about my research now . What do you think ?

#2 gebirgsziege

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Posted 19 October 2009 - 03:31 AM

We are having not much space and only two rooms for everything (one for electrophoresis and one for the rest)....you have to be careful working and invest some more time in cleaning your equipment but it works fine. Always include positve and negative controls in every single experiment (so every PCR you run, every DNA extraction, every gel you run etc.)
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 :D 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.

A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#3 Clare

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Posted 19 October 2009 - 04:44 AM

I have routinely set up everything, and ran all gels in the same room. In my first lab, that was 4 years. And I never had problems with contamination. I have done the same in my current lab (2.5 years).
Clare

#4 phage434

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Posted 19 October 2009 - 04:59 AM

We've been doing PCRs in the same room for many, many years. I think the issue is what you are doing with the PCR, rather than anything else. If you are trying to use PCR to detect the presence of a specific fragment at low concentrations, then I believe you may have an issue. If you are doing routine cloning and amplification to isolate a gene, e.g., then I don't believe there is a problem. I've done thousands of colony pcr reactions, where each was attempting to determine if a specific fragment was present, and have had no trouble with cross contamination. If I were running a forensics lab and wanted to check for DNA on a particular item, then I would be very very concerned.
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.

#5 Bassaml7

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Posted 19 October 2009 - 08:53 AM

Thank you all . In fact my research involves the detection of SNP using the Amplification refractory mutation system method ( ARMS ) , also called Allele specific amplification method . My mission is to determine is to screen the population to determine a certain SNP percentage . So it involves a lot of different samples and amplifications so there is no way to tolerate cross-contamination mistakes. Any contamination could lead into a false percentage.

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.


#6 HomeBrew

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Posted 19 October 2009 - 01:15 PM

We, too, have been doing all aspects of PCR in a single room for many years and uncounted thousands of reactions, with no difficulties (and no filtered tips, either...) :lol:.

#7 fishdoc

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Posted 19 October 2009 - 02:52 PM

Set up all conventional PCR reactions at my benchtop, then PCR in another room, which also contains the gel rigs. For qPCR, set up everything under a hood. The RT step is done in the same room as the thermocyclers and electrophoresis equipment, but the qPCR reaction is done in a thermocycler in a core facilities lab.

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.

#8 Bassaml7

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Posted 19 October 2009 - 10:22 PM

Thank you all ...

#9 dtae

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Posted 06 March 2011 - 12:39 PM

Bassaml7 - out of curiosity, did you end up having any problems with contamination?

#10 Maddie

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Posted 10 March 2011 - 08:58 AM

Gosh, guys, you are gonna give me nightmares :lol: .
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.
Good luck.
Maddie
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




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