IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-25: Difference between revisions
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Digested for 6 hours at 45C. | Digested for 6 hours at 45C. | ||
== Restreak and colony PCR == | |||
I selected, restreaked, colony PCR'd, and inoculated 20 colonies from Nick's ligation #5 that he plated on 2006-8-24. | |||
Ligation conditions: | |||
*KaiC Gel + Gel Backbone #5 II at 17C |
Revision as of 13:48, 25 August 2006
Miniprep of Nick's cultures #5 and #8
I miniprepped the 4 cultures Nick inoculated last night, and labeled them 5-1, 5-2, 8-1, and 8-2.
From Nick's email:
All three colonies are KaiB constructs. I have made 4 overnight
cultures with the following labels:
"NES 5" is a putative KaiB insert in a J04500 from a 6:1 Insert:Backbone
reaction using the NEB ligase.
"NES 5" is a second culture started from the same colony.
"NES 8" is a putative KaiB insert in a J04500 from a 3:1 Insert:Backbone
reaction using the Roche ligase.
"NES 8" is from a different (*not* clonal) colony from the same ligation
reaction as the other NES 8.
Diagnostic digests of Nick's cultures #5 and #8
Digest # | Plasmid | Enzymes |
1 | 5-1 | X |
2 | 5-1 | X, P |
3 | 8-1 | X |
4 | 8-1 | X, P |
5 | 8-2 | X |
6 | 8-2 | X, P |
\X digest reactions
- 19.5 µL DNA
- 2.25 µL H2O
- 2.5 µL NEBuffer 2
- 0.5 µL XbaI
- 0.25 µL 100x BSA
\X digest master
- 9 µL H2O
- 10 µL NEBuffer 2
- 2 µL XbaI
- 1 µL 100x BSA
Pipette 5.5 µL into each reaction.
\X-P digest reactions
- 19.5 µL DNA
- 1.75 µL H2O
- 2.5 µL NEBuffer 3
- 0.5 µL XbaI
- 0.5 µL PstI
- 0.25 µL 100x BSA
\X-P digest master
- 7 µL H2O
- 10 µL NEBuffer 3
- 2 µL XbaI
- 2 µL PstI
- 1 µL 100x BSA
Pipette 5.5 µL into each reaction.
Digested for 6 hours at 45C.
Restreak and colony PCR
I selected, restreaked, colony PCR'd, and inoculated 20 colonies from Nick's ligation #5 that he plated on 2006-8-24.
Ligation conditions:
- KaiC Gel + Gel Backbone #5 II at 17C