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    • #17425
      bravebeaker
      Participant

      Hi all,
      Recently I have designed the following primers to amplify a part of the promoter region of my gene of interest.

      fw: 5′- gggg CCGCGG TG AGC AAG TAT ACC AAC CAT -3′ (Sacii) Tm 63.3
      rv: 5′- gggg TCTAGA CAA TGT ACA CCA ACA TAT AC -3′ (Xbai) Tm 56.5

      I am using genomic DNA as template and KOD kit.
      Unfortunately I have been unsuccessful in amplifying the target region.

      I have tried many annealing temperatures such as 50 up to 60.

      Any idea how to solve this problem? If the primers are of no use, how can I redesign them? I need the SacII and XbaI sites.

      Thank you a lot.

    • #114063
      JackBean
      Participant

      The difference of Tms is pretty big, 7°C! You can either try knock-down PCR or try different primers. But remember, that you need to check Tm of only the part which will bind to the gDNA. Why do you have 4 guanosines in the beginning, that seems like way too much. Both your enzymes are fine with only 1 base in the end: https://www.neb.com/tools-and-resources … -fragments

    • #114067
      bravebeaker
      Participant

      Thank you for the help and the link. I am going to give it another try if not I am going to redesign the the Fw primer.
      I calculated the Tm by using the 2+4 rule but it seems its not very accurate.

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