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Team:Cambridge/LabBook/Week8
From 2010.igem.org
Contents |
Monday
Result (from Expt. 68):
No growth overnight, 2 growths out of 3 plates (+1 fungus) after weekend of growing. No glow.
69. Expt: Growing up of cell cultures of above experimental results & of results on p49 (Will)
Strains G1, j1 and G28wh were grown up in 5ml LB + 2µl Chloramphenicol, and on plates containing LB+Cm.
Left to grow at 30°C for 24h.
Results: Strains grew but did not glow after 48h.
70. Expt: Miniprep pSB1C3 from colonies (Anja)
Suspended a 'streak' of bacteria transformed with BBa_J04450 (registry part in pSB1C3) in 250µl buffer P1. Followed 'Bench protocol: Qiagen Spin Miniprep Kit Using a Microcentrifuge'.
Nanodrop 12.8ng/µl
71. Expt: Gibson assembly of pBAD, luxCD,AB,EG and pSB1C3 (Anja)
PCR:
| Template | Primer f. | Primer r. | Amplified Fragment |
| I0500 | prefix.f.pBadstart | luxCstart.r.pBADend | pBAD (A) |
| pJS555 | pBADend.f.luxCstart | luxAstart.r.luxDend | luxCD (B) |
| pJS555 | luxDend.f.luxAstart | luxEstart.r.luxBend | luxAB (C) |
| pJS555 | luxBend.f.luxEstart | suffix.rev.luxGend | luzEG (D) |
| BBa_J04450 | luxGend.for.suffix | pBADstart.r.prefix | pSB1C3 (E) |
BBa_J04450: once purified (E), once colony PCR (EC)
Followed protocol on p38 for PCR mixtures and programs.
Gel electrophoresis
Self-cast 1% agarose gel loaded as follows:
| Easyladder II | A | A | B | B | C | C | D | D | E | E | EC | EC |
| 10µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl | 17µl |
Run at 120V.
No bands were visible. Not even in the marker lane. Since a gel was that had been sitting on the bench for a few days and SYBR Safe Dye is light sensitive it is assumed that the dye had become non-functional.
A 1% Agarose gel was loaded as follows:
| Easyladder II | A | A | B | C | D | E? | EC? | |
| 10µl | 12µl | 12µl | 12µl | 12µl | 12µl | 12µl | 12µl | PCR Rxn |
| 3µl | 3µl | 3µl | 3µl | 3µl | 3µl | 3µl | 6x LD | |
| 5µl | 5µl | 5µl | 5µl | 5µl | 5µl | 5µl | Nuclease-free H20 |
