Team:Washington/Notebook/SOEingPCR

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%
 * 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
 * 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