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Team:Colombia/Notebook/Digestions
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| + | '''Double Digestions''' | ||
| - | + | 2 hour incubation (37°C) followed by an hour incubation period at room temperature and another 2 hours at 37°C. | |
We used serial digestions to improve SpeI efficiency since it wasn’t working correctly as follows: | We used serial digestions to improve SpeI efficiency since it wasn’t working correctly as follows: | ||
Latest revision as of 23:09, 28 September 2011
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Digestion Protocols
Double Digestions
2 hour incubation (37°C) followed by an hour incubation period at room temperature and another 2 hours at 37°C.
We used serial digestions to improve SpeI efficiency since it wasn’t working correctly as follows:
| Ultrapure water | 23.8ul |
| Promega ® Buffer D | 4ul |
| XbaI | 0.6uL |
| EcoRI*/PstI** | 0.6uL |
| DNA | 1ug |
| Final volume | 40uL |
We then purified the digestions using the Promega ® Wizard Sv and PCR Clean-Up system which were then eluted to 17.6 uL of ultrapure water or precipitated using Sodium Acetate and ethanol in ice.
| Promega ® Buffer H | 2uL |
| EcoRI**/PstI* | 0,6uL |
| DNA | 17,6uL |
| Final volume | 20uL |
[*] Plasmid opening [**] Fragment extraction
Run a 1% agarose electrophoresis for 55 minutes with 70V, and purify the expected weight bands using the Promega ® Wizard Sv and PCR Clean-Up system.
