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Team:Washington/Notebook/SOEingPCR
From 2009.igem.org
(Difference between revisions)
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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: | + | **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% | ||
Revision as of 02:07, 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%
