Difference between revisions of "GGA for BsmBI"
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Macampbell (talk | contribs) (Created page with "We are trying to clone two gBlocks designed with sticky ends after digestion with BsmBI. '''Thermocycle Program''' * 20 minutes at 55 C (start with digestion cocktail below)...") |
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** 1 min 55 C | ** 1 min 55 C | ||
** (end loop) 20 cycles total | ** (end loop) 20 cycles total | ||
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* hold at 21 C | * hold at 21 C | ||
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Revision as of 19:01, 10 November 2014
We are trying to clone two gBlocks designed with sticky ends after digestion with BsmBI.
Thermocycle Program
- 20 minutes at 55 C (start with digestion cocktail below)
- pause to put in 5 µL ligation mixture (see below)
- 5 minutes at 16 C
- (start loop below)
- 2 min 16 C
- 1 min 55 C
- (end loop) 20 cycles total
- hold at 21 C
Digestion cocktail
- water 6.5 µL
- 25 ng vector cut with EcoRI and Spe (gel purified) (1.76 µL)
- 3x molar insert 1 (P1) 1 µL
- 3x molar insert 2 (P2) 1 µL
- 10x cutsmart buffer 1 µL
- BsmBI enzyme 0.5 µL
- final volume 10 µL
Ligation mixture
- water 3 µL
- 10X ligation buffer 1.5 µL
- ligase 0.5 µL