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Team:Cambridge/Notebook/12
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Week 11: Monday 27th September - Sunday 3rd October
113. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)
- Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
- Nanodrop readings:
| Nanodrop reading (ng/µl) | |
| Cm+PP+pBAD+pSB1C3 | 48.5 |
| YFP+rbs | 74.8 |
| RFP+rbs | 32.9 |
| CFP+rbs | 46.7 |
pSB1C3 from freezer was used at 12.9ng/µl
- Restrict using protocol on p88, using following quantities:
| Cm+PP+pSB1C3 | YFP (for PP) | YFP (for pBAD) | RFP (for PP) | RFP (for pBAD) | CFP (for PP) | CFP (for pBAD) | pSB1C3 | |
| Nuclease-free H20 | 1 | 6 | 6 | 1 | 1 | 6 | 6 | 1 |
| 10x FD Buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Plasmid DNA | 15 | 10 | 10 | 15 | 15 | 10 | 10 | 16 |
| EcoRI | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| SpeI | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 0 |
| XbaI | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 |
| PstI | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| * | * | * | * |
- We ran this gel on an E-gel and realised that those lanes with '*' had been cut with the wrong enzymes - PP+pSB1C3 should have been cut with S+P, the RFPs should have been cut with X+P.
- We carried on with those that had worked with just pBAD.
See continuation below.
114. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD
Arabinose concentration varying from 0µM to 10mM
- 1. 0µM: 0Ara/30µl of H20
- 2. 1µM: 1µl of Ara@100µM/29µl of H20
- 3. 3µM: 3µl of Ara@100µM/27µl of H20
- 4. 10µM: 10µl of Ara@100µM/20µl of H20
- 5. 30µM: 30µl of Ara@100µM/no H20
- 6. 100µM: 1µl of Ara@10mM/29µl of H20
- 7. 300µM: 3µl of Ara@10mM/27µl of H20
- 8. 1mM: 10µl of Ara@10mM/20µl of H20
- 9. 3mM: 30µl of Ara@10mM/no H20
- 10. 10mM: 1µl of Ara@1M/29µl of H20
- 11. No luciferin, 100µM Ara => 1µl of 10mM, 30.5 of H20
D-luciferin at 100µM -> 15µl.
Total volume 100µl per well. 60.5µl of overnight culture
Plate layout:
| 1-3 | 4-6 | 7-9 | 10-12 | |
| A | PP(1) | PP(2) | PP(3) | PP(4) |
| B | PP(5) | PP(6) | PP(7) | PP(8) |
| C | PP(9) | PP(10) | PP(11) | LC(5) |
| D | ||||
| E | ||||
| F | LC(1) | LC(6) | LC(3) | LC(4) |
| G | LC(7) | LC(8) | ||
| H | LC(9) | LC(10) | LC(11) | Blanks |
µl°C
