Team:Washington/Notebook/SOEingPCR

SOEing PCR

1. Design primers

• • VF2 / VR = standard forward and revers primer from original construct

1. 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. 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. 1. T7F+M1-R
   2. M1-F+M2-R
   3. M2-F+M3-R
   4. M3-F+T7R

• • PCR 1 Master Mix

1. 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. 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. 1. 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. 1. 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. 1. 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. 1. 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