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QC & Lab standards - PCR

  • 16-09-2009 4:54pm
    #1
    Phototoxin
    Closed Accounts Posts: 1,398 ✭✭✭


    I have a question....

    In DNA PCR;

    If a sample read 0 in quantification it can be re-run in PCR in the hopes of getting a profile.

    However -ve control standards in this case would not be run. IE control = 0 = :-)

    Is this a case of double standards thought? - knowing that a 0 quantified result with 0 profile from PCR can be re-run and a potential result obtained?


Comments

  • #2
    Kevster
    Closed Accounts Posts: 2,980 ✭✭✭Kevster


    Hi,

    Negative controls should always be run, irrespective of what the spectrophotometer gives as it's reading for quantification. If there are no negative controls, then the results are unreliable. UV/Visible spectrophotometers are only sensitive down to nanoMolar concentrations (I think), but there could be minute amounts of DNA that are out of this range; and these 'out of range' DNA samples could be amplified in the PCR. Thus, a 0 quantification result could become a positive in the actual PCR reaction.

    ALWAYS USE NEGATIVE CONTROLS

    Kevin


  • #3
    Phototoxin
    Closed Accounts Posts: 1,398 ✭✭✭Phototoxin


    sorry what I mean is that a zero scoring (passed) control would not be run but a zero scoring sample (failed) would be as it is possible that a zero score sample can still yeild a profile yet each time you get a passed control you don't second guess..

    I hope that makes more sense


  • #4
    Kevster
    Closed Accounts Posts: 2,980 ✭✭✭Kevster


    Hmm, I think I know what you mean now; but if any sample fails then that's pretty much it. If you run it again on it's own, then it could technically pick up contamination in between runs; and you'd have to run the controls again anyway with it.

    Look at it this way: Do you think that your approach would be looked upon favourably in a legal case? ... ...I don't think so. Let me know what you think.


  • #5
    Phototoxin
    Closed Accounts Posts: 1,398 ✭✭✭Phototoxin


    Well I would think that the fact that the 0 result in a sample is second guessed opens up the possiblity for a 0 result in a -ve control to be second guessed also.


  • #6
    Kevster
    Closed Accounts Posts: 2,980 ✭✭✭Kevster


    That depends on how many samples you're actually running. Is it just one sample and one control? If so - and if the sample gets a 0 - then by all means repeat the experiment, but from scratch, and using a new PCR mix (if that's possible). You can still just repeat it for the old sample unoficially.

    I used to run 90+ samples at any one time, which is why I was at odds with what you were saying.


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