Team:Washington/Notebook/SOEingPCR
From 2009.igem.org
SOEing PCR
- Design primers
- VF2 / VR = standard forward and revers primer from original construct
- 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%
- 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%
- 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
- An example assembly altering DNA at three different points (can do this with as many or as few as needed for your application)
- T7F+M1-R
- M1-F+M2-R
- M2-F+M3-R
- M3-F+T7R
- PCR 1 Master Mix
- 36.5uL dH2O
- 10uL Phusion Buffer
- 1uL plasmid template
- 1uL 25mM dNTPs
- 0.5uL 100uM Forward primer
- 0.5uL 100uM Reverse primer
- 0.5uL Phusion
- PCR 1 Cycle
- 98C, 30sec
- 98C, 10sec
- 58C, 10sec
- 72C, 30sec/kb
- Repeatsteps2-4 29x
- 72C, 5min
- 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 notPCR 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 corect length. Change annealing temperature of PCR to gradient (55-72)
- Single band at correct fragment size. PCR purify all of PCR product and spec
- possible outcomes
- Run DNA gel (5uL PCR reaction + 1uL 6x loading dye)