Team:Washington/Notebook/NheI PstI


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Gene Assembly using the NheI and PstI sites

  1. Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
  2. Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
  3. Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
  4. Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to that of VR
  5. Amplify your gene using the designed forward oligo and VR
  6. PCR purify the PCR product
    1. To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
  7. Digest PCR product with XbaI and PstI
    1. PCR purify
  8. Digest Vector with NheI and PstI
    1. PCR purify
  9. Mix insert and vector in 3:1 ratio and ligate
  10. Transform into competent cells
  11. Screen cells for correct insert using VF2 and VR