Team:Washington/Notebook/SOEingPCR

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SOEing PCR

  1. Design primers
    • VF2 / VR = standard forward and revers primer from original construct
    1. For mutations/insertions/deletions of 1-15 base pairs
    • Forward: 5'-----------------------XXXX-----------------3'
    • Template: 5'----------------------------------------------3'
    • Reverse: 3'-----------------------XXXX-----------------5'
      • X = mismatch base pair with template
      • - = Matching base pair with template. Make sure that 15-25 base pairs match. The Tm of the matching regions should be >60C, and the GC content 40%-60%
    1. For mutations/insertions/deletions of 15+ base pairs
    • Forward: ..............................................5'-XXXX-----------------3'
    • Template:..5'------------------------------------------------3'
    • Reverse:...3'-----------------------XXXX-5'
      • X = mismatch base pair with template
      • - = Matching base pair with template. Make sure that 15-25 base pairs match. The Tm of the matching regions should be >60C, and the GC content 40%-60%
  1. PCR: Adding the mutations
    • An example assembly altering DNA at three different points (can do this with as many or as few as needed for your application)
      • Top Strand..... T7F........M1-F..................M2-F....................M3-F...................
      • Ref Sequence ...................X.........................X...........................X.....................
      • Bottom Strand ...............M1-R..................M2-R....................M3-R............T7R
      • MX-F = forward mutation primer X
      • MX-R = reverse mutation primer x
      • X = mutation site
      • ..... = place holder
    • Samples, 1 set without DMSO, 1 set with 5% DMSO
    1. T7F+M1-R
    2. M1-F+M2-R
    3. M2-F+M3-R
    4. M3-F+T7R
    • PCR 1 Master Mix
    1. 36.5uL dH2O
    2. 10uL Phusion Buffer
    3. 1uL plasmid template
    4. 1uL 25mM dNTPs
    5. 0.5uL 100uM Forward primer
    6. 0.5uL 100uM Reverse primer
    7. 0.5uL Phusion
    • PCR 1 Cycle
    1. 98C, 30sec
    2. 98C, 10sec
    3. 58C, 10sec
    4. 72C, 30sec/kb
    5. Repeatsteps2-4 29x
    6. 72C, 5min
    7. 4C, forever
    • Run DNA gel (5uL PCR reaction + 1uL 6x loading dye)
      • possible outcomes
        • Single band at correct fragment size. PCR purify all of PCR product and spec
          • Do not PCR purify chunks less that 70bp, estimate concentration based on gel
        • Multiple bands.Run another gel using all PCR product, gel purify correct band, spec
        • No bands / no bands at correct length. Change annealing temperature of PCR to gradient (55-72)
  1. PCR 2: Putting the pieces together
    • Need equal-molar concentrations of each piece,normalize concentration to size
    • Make 20uL mix of the pieces
    • If DMSO worked in PCR 1, add 5% DMSO in PCR 2
    • PCR 2 Master Mix
      1. 18.5uL dH2O
      2. 20uL of pieces mix
      3. 10uL of Phusion Buffer
      4. 1uL 25mM dNTPs
      5. 0.5uL Phusion
    • PCR 2 cycle
      1. 98C, 30sec
      2. 98C, 10sec
      3. 58C,10sec (or same as PCR 1)
      4. 72C, 30sec/kb
      5. Repeat steps 2-4 5x
      6. 72C, 5min
      7. 4C, forever
    • No need to purify or anything after PCR 2, just go directly to PCR#3. (I have PCR purified after this step (do not gel purify) and had everything still work though.)
  1. PCR 3: Amplifying full length construct
    • If DMSO worked in PCR 1, add DMSO 5% in PCR 3
    • PCR 3 Master Mix
      1. 36.5uL dH2O
      2. 10uL Phusion Buffer
      3. 1uL PCR 2 product
      4. 1uL 25mM dNTPs
      5. 0.5uL 100uM T7F
      6. 0.5uL 100uM T7R
      7. 0.5uL Phusion
    • PCR 3 cycle
      1. 98C, 30sec
      2. 98C, 10sec
      3. 58C,10sec (Tm for T7)
      4. 72C, 30sec/kb
      5. Repeat steps 2-4 29x
      6. 72C, 5min
      7. 4C, forever
    • Run gel
      • Possible outcomes
        • Single band at correct fragment size, PCR purify your final product
        • Multiple bands, run another gel using all PCR product, gel purify correct band
        • No bands or no bands at right size, Change annealing temperature of PCR to gradient (55-72)
  1. Next Step: Standard cloning
    • digest,purify,ligate,transform
    • Happy Dance