Difference between revisions of "GGA for BsmBI"
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'''Thermocycle Program''' | '''Thermocycle Program''' | ||
* 20 minutes at 55 C (start with digestion cocktail below) | * 20 minutes at 55 C (start with digestion cocktail below) | ||
| − | |||
* 5 minutes at 16 C | * 5 minutes at 16 C | ||
| − | * (start loop below) | + | * push pause when it reaches 16 C |
| + | * put in 5 µL ligation mixture (see below) | ||
| + | * push pause again to resume 5 minute timer | ||
| + | * (when 5 minute timer is done, start loop below) | ||
** 2 min 16 C | ** 2 min 16 C | ||
** 1 min 55 C | ** 1 min 55 C | ||
** (end loop) 20 cycles total | ** (end loop) 20 cycles total | ||
| − | * hold at | + | * hold at 16 C |
<br> | <br> | ||
<hr> | <hr> | ||
Latest revision as of 15:36, 18 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)
- 5 minutes at 16 C
- push pause when it reaches 16 C
- put in 5 µL ligation mixture (see below)
- push pause again to resume 5 minute timer
- (when 5 minute timer is done, start loop below)
- 2 min 16 C
- 1 min 55 C
- (end loop) 20 cycles total
- hold at 16 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
