IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-14: Difference between revisions
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It appears that our vector self-ligated. | It appears that our vector self-ligated. | ||
==Digestion Assay for 8/14== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''Label''' | |||
| align="center" style="background:#f0f0f0;"|'''DNA''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
| align="center" style="background:#f0f0f0;"|'''Water''' | |||
| align="center" style="background:#f0f0f0;"|'''Enzymes''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
| align="center" style="background:#f0f0f0;"|'''NEB buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''BSA (100x)''' | |||
| align="center" style="background:#f0f0f0;"|'''Time''' | |||
| align="center" style="background:#f0f0f0;"|'''Master''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|-} |
Revision as of 12:41, 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 |