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Thread: A quantitative real-time PCR method for absolute telomere length

  1. #1 A quantitative real-time PCR method for absolute telomere length 
    New Member
    Join Date
    May 2014
    I am curently trying to determine the absolute telomere length by using "Cawthon's quantitative real-time - Biotechniques, 2008". My problem arrise by the primer dimer formation in my negative control well as well in the formation of the standard curve. I am using a " StepOne Real-Time PCR System" with "rapid SeniFast SYBR Hi-Rox kit". AND telo F/R AND tel 1/2 primers, my condition according to the publication : and 3step hot start RT PCR with:

    1 95 C 2min

    40 95 C 5s
    61 C 10s
    72 C 15s

    and meltin curve.

    Everything i tried till now seem to form primer dimers. can someone help withough having to order new primers if possible??
    ps i also forgot to mention that concetration of telomere oligo in standard cure sets off 1/10 dilutions with highest 60pg.
    I was assuming that it was a contamination thus by replicating the experiment , researching online and changing varies of factors i realise it was primer dimer formation. it was hard to diferenciate as sample was extremely low concetration.

    Please if needed any more information about my conditions, don not hesitate to ask.
    Thanks in advance

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