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Team:Washington/Notebook/SOEingPCR
From 2009.igem.org
(Difference between revisions)
| Line 5: | Line 5: | ||
##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: 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% | ||
##For mutations/insertions/deletions of 15+ base pairs | ##For mutations/insertions/deletions of 15+ base pairs | ||
| - | **Forward: ............................................'''5'-XXXX-----------------3'''' | + | **Forward: ..............................................'''5'-XXXX-----------------3'''' |
**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 14:04, 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%
