IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-14: Difference between revisions
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Add 5.5 µL of the corresponding master to each tube. | Add 5.5 µL of the corresponding master to each tube. |
Revision as of 12:52, 14 August 2006
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To-do
- Midiprep pSB4A, J04500 (online instructions here, page 16)
- Insert:
- Digest miniprepped KaiA/B/C in GeneArt vector with X-P
- Backbone:
- Digest J04500\S-P, \X-P (for Stage I constructs and BB high-copy)
- Find and transform B0034
- KaiABC ligation:
- KaiABC\X-P + J04500\S-P (stage I)
- KaiABC\X-P + pSB1AK3 (well-formed BB)
- KaiABC\X-P + B0034\S-P (stage I)
- Sequencing:
- Sequence miniprepped KaiA/B/C + GA vectors, natural KaiA
- Western blots:
- Ligate GFP dev and pSB4A3
- Inoculate GFP dev + pSB1A2
E-gel images of colony PCR of J04500 + KaiA/B/C
It appears that our vector self-ligated.
Digestion Assay for 8/14
Label | DNA | Amount | Water | Enzymes | Amount | NEB buffer | BSA (100x) | Time | Master | Total | |
Digests | 1 | J04500 | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 2 hrs | 1 | 25 µL |
2 | J04500 | 19.5 µL | 1.75 µL | SpeI & PstI | 0.5 µL, 0.5 µL | Buffer 2 (2.5 µL) | 0.25 µL | 2 hrs | 2 | 25 µL | |
3 | J04500 | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 4 hrs | 1 | 25 µL | |
4 | J04500 | 19.5 µL | 1.75 µL | SpeI & PstI | 0.5 µL, 0.5 µL | Buffer 2 (2.5 µL) | 0.25 µL | 4 hrs | 2 | 25 µL | |
5 | KaiA | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 2 hrs | 1 | 25 µL | |
6 | KaiB | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 2 hrs | 1 | 25 µL | |
7 | KaiC | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 2 hrs | 1 | 25 µL | |
8 | KaiA | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 4 hrs | 1 | 25 µL | |
9 | KaiB | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 4 hrs | 1 | 25 µL | |
10 | KaiC | 19.5 µL | 1.75 µL | XbaI & PstI | 0.5 µL, 0.5 µL | Buffer 3 (2.5 µL) | 0.25 µL | 4 hrs | 1 | 25 µL | |
Master Mixes | 1 | - | - | 15.75 µL | XbaI & PstI | 4.5 µL, 4.5 µL | Buffer 3 (22.5 µL) | 2.25 µL | - | - | 49.5 µL |
2 | - | - | 7 µL | SpeI & PstI | 2 µL, 2 µL | Buffer 2 (10 µL) | 1 µL | - | - | 22 µL |
Add 5.5 µL of the corresponding master to each tube.