Team:Washington/Notebook/SOEingPCR
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+ | __NOTOC__ | ||
+ | {{Template:Team:Washington/Templates/Header}} | ||
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====SOEing PCR==== | ====SOEing PCR==== | ||
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**VF2 / VR = standard forward and revers primer from original construct | **VF2 / VR = standard forward and revers primer from original construct | ||
##For mutations/insertions/deletions of 1-15 base pairs | ##For mutations/insertions/deletions of 1-15 base pairs | ||
- | **Forward: 5'-----------------------XXXX-----------------3' | + | **Forward: '''5'-----------------------XXXX-----------------3'''' |
- | **Template: | + | **Template: ''' 5'----------------------------------------------3'''' |
- | **Reverse: | + | **Reverse: ''' 3'-----------------------XXXX-----------------5'''' |
- | ***X = mismatch base pair with template | + | ***'''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% | + | ***'''-''' = 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 | ##For mutations/insertions/deletions of 15+ base pairs | ||
- | **Forward: | + | **Forward: ..............................................'''5'-XXXX-----------------3'''' |
- | **Template: | + | **Template:..'''5'------------------------------------------------3'''' |
- | **Reverse: | + | **Reverse:...'''3'-----------------------XXXX-5'''' |
- | ***X = mismatch base pair with template | + | ***'''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% | + | ***'''-''' = 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 | ||
+ | ##'''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 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) | ||
+ | #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 | ||
+ | ###18.5uL dH2O | ||
+ | ###20uL of pieces mix | ||
+ | ###10uL of Phusion Buffer | ||
+ | ###1uL 25mM dNTPs | ||
+ | ###0.5uL Phusion | ||
+ | **PCR 2 cycle | ||
+ | ###98C, 30sec | ||
+ | ###98C, 10sec | ||
+ | ###58C,10sec (or same as PCR 1) | ||
+ | ###72C, 30sec/kb | ||
+ | ###Repeat steps 2-4 '''5'''x | ||
+ | ###72C, 5min | ||
+ | ###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.) | ||
+ | #PCR 3: Amplifying full length construct | ||
+ | **If DMSO worked in PCR 1, add DMSO 5% in PCR 3 | ||
+ | **PCR 3 Master Mix | ||
+ | ###36.5uL dH2O | ||
+ | ###10uL Phusion Buffer | ||
+ | ###1uL PCR 2 product | ||
+ | ###1uL 25mM dNTPs | ||
+ | ###0.5uL 100uM T7F | ||
+ | ###0.5uL 100uM T7R | ||
+ | ###0.5uL Phusion | ||
+ | **PCR 3 cycle | ||
+ | ###98C, 30sec | ||
+ | ###98C, 10sec | ||
+ | ###58C,10sec (Tm for T7) | ||
+ | ###72C, 30sec/kb | ||
+ | ###Repeat steps 2-4 29x | ||
+ | ###72C, 5min | ||
+ | ###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) | ||
+ | #Next Step: Standard cloning | ||
+ | **digest,purify,ligate,transform | ||
+ | **Happy Dance | ||
+ | |||
+ | {{Template:Team:Washington/Templates/Footer}} |
Latest revision as of 00:47, 17 October 2009
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 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)
- 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)
- 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
- 18.5uL dH2O
- 20uL of pieces mix
- 10uL of Phusion Buffer
- 1uL 25mM dNTPs
- 0.5uL Phusion
- PCR 2 cycle
- 98C, 30sec
- 98C, 10sec
- 58C,10sec (or same as PCR 1)
- 72C, 30sec/kb
- Repeat steps 2-4 5x
- 72C, 5min
- 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.)
- PCR 3: Amplifying full length construct
- If DMSO worked in PCR 1, add DMSO 5% in PCR 3
- PCR 3 Master Mix
- 36.5uL dH2O
- 10uL Phusion Buffer
- 1uL PCR 2 product
- 1uL 25mM dNTPs
- 0.5uL 100uM T7F
- 0.5uL 100uM T7R
- 0.5uL Phusion
- PCR 3 cycle
- 98C, 30sec
- 98C, 10sec
- 58C,10sec (Tm for T7)
- 72C, 30sec/kb
- Repeat steps 2-4 29x
- 72C, 5min
- 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)
- Possible outcomes
- Run gel
- Next Step: Standard cloning
- digest,purify,ligate,transform
- Happy Dance