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Team:Paris Liliane Bettencourt/Notebook/2011/08/04/
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==Danyel & Camille== | ==Danyel & Camille== | ||
| - | ===T7 amber== | + | ===T7 amber=== |
According to the T7 amber sequencing results the S15 strain only held the terminator and not the T7 polymerase. | According to the T7 amber sequencing results the S15 strain only held the terminator and not the T7 polymerase. | ||
The cloning step of the T7 + standard terminator has to be redone. | The cloning step of the T7 + standard terminator has to be redone. | ||
Revision as of 16:28, 4 August 2011
Contents |
Cyrille
Elimitation of the EcoRI site by the EcoRI-Klenow method
The template will overgo a menaged digestion with normal EcoRI. 12 tubes of 2 µg of DNA template will be prepared, in the fastdigest buffer. They will be digested with EcoRI for different duration: 0 - 1 - 3 - 5 - 6 - 7 - 8 - 9 - 10 - 11 - 13 - 15 min
The inactivation of the enzyme is done by eating 5 min at 80°C.
The results are loaded on a gel.
The bands 3-5 are cutted together, as well as 6,7,8,9, and 10-11. They will undergo a gel extraction.
The extermities are blunted using Taq polymerase on a PCR machine.
Danyel & Camille
T7 amber
According to the T7 amber sequencing results the S15 strain only held the terminator and not the T7 polymerase. The cloning step of the T7 + standard terminator has to be redone.
