Colony PCR

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    • #10632
      shivakumar
      Participant

      I did a colony pcr reaction (final vol 100ul) and the product was electrophoresed in 1.2% agarose gel . After complete run I observed a smear from one end to another end in each well (of the gel) including blank (without template).
      I thought that problem with concentration of DNA template and tried in other trials by reducing the concentration of the template. But I got the same result.
      To overcome the contamination problem I tried with new components, new plastic ware and auticlaved glass ware
      but the result is same.
      Can anybody suggest me to overcome this problem?

      This is the set up I performed

      10X Taq Buffer-10ul
      10mM dNTPS -2 ul
      M13F primer(3pmol/ul)-2ul
      M13R Primer (3pmol?ul)-2ul
      Taq- 1ul
      Template-usually 4 ul
      Sterile water-79ul

      PCR Programme
      94c-5′
      94c-1′
      55c-1:30′
      72c-1′
      30 cycles
      72c-10

    • #87939
      mith
      Participant

      You’ve changed everything, but it’s still in your blank? Then it suggests your changed stuff is still contaminated…find a lab with working results and see if they will let you borrow their stuff. If that still doesn’t work, then it sounds like your protocol is contaminating it.

    • #88687
      pcrboy
      Participant

      determining where the smear appears may help determine whether it is chromosome DNA or plasmid DNA.

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