M13 redesign, expt'l details

User:Nkuldell:M13 redesign

Vectors

M13K07 plasmid map, from NEB

• M13KE [[1]]
• pUC118 [[2]] or 119 [[3]]
• M13K07 (helper phage)[|sequence][[4]]

File:M13KO7 zerocutters.txt
File:M13KO7 singlecutters.txt
File:M13KO7 2cutters.txt

Primers

For M13K07 manipulations

• First need to delete Pst site from M13K07 polylinker

these oligos should both destroy Pst and add unique EcoRI to position 8083

• NO133: ChangePst2RI_top
  
  • 5'ATAG AAT TCT ATG CA
• NO134: ChangePst2RI_btm
  
  • 5'ACG TTAT CTT AAG AT
    

• Next step is to add PstI to Petrenko site

use site directed mutagenesis to change GCT to GCA (silent change at Ala, first residue in mature protein)

• NO135: AddPst2gVIII_fwd
  
  • CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC
  • Tm 74°
• NO136: AddPst2gVIII_rev
  • GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G
  • Tm 74°

For M13K07 sequencing

• NO137: M13KO7_seq1100_fwd
  • TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC
  • Tm 65°