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Team:Washington/Notebook/SOEingPCR
From 2009.igem.org
(Difference between revisions)
| Line 13: | Line 13: | ||
**Template:..'''5'------------------------------------------------3'''' | **Template:..'''5'------------------------------------------------3'''' | ||
**Reverse:...'''3'-----------------------XXXX-5'''' | **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% |
Revision as of 14:09, 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%
