"
Team:Washington/Notebook/SOEingPCR
From 2009.igem.org
(Difference between revisions)
| Line 19: | Line 19: | ||
***Top Strand..... '''T7F'''........'''M1-F'''..................'''M2-F'''....................'''M3-F'''................... | ***Top Strand..... '''T7F'''........'''M1-F'''..................'''M2-F'''....................'''M3-F'''................... | ||
***Ref Sequence ...................'''X'''.........................'''X'''...........................'''X'''..................... | ***Ref Sequence ...................'''X'''.........................'''X'''...........................'''X'''..................... | ||
| - | ***Bottom Strand ...............'''M1-R'''..................'''M2-R'''....................'''M3-R'''............T7R | + | ***Bottom Strand ...............'''M1-R'''..................'''M2-R'''....................'''M3-R'''............'''T7R''' |
***'''MX-F''' = forward mutation primer '''X''' | ***'''MX-F''' = forward mutation primer '''X''' | ||
***'''MX-R''' = reverse mutation primer '''x''' | ***'''MX-R''' = reverse mutation primer '''x''' | ||
***'''X''' = mutation site | ***'''X''' = mutation site | ||
***..... = place holder | ***..... = place holder | ||
Revision as of 14:24, 16 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
- An example assembly altering DNA at three different points (can do this with as many or as few as needed for your application)
