Team:Cambridge/LabBook/Week8

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(New page: <div align="center">Week 8: Monday 30th August - Sunday 5th September</div> ==Monday== Result (from Expt. 68): No growth overnight, 2 growths out of 3 plates (+1 fungus) after weekend o...)
Line 54: Line 54:
BBa_J04450: once purified (E), once colony PCR (EC)
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:
 +
{|class="wikitable"
 +
|-
 +
|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:
 +
{|class="wikitable"
 +
|-
 +
|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
 +
|}

Revision as of 09:03, 24 September 2010

Week 8: Monday 30th August - Sunday 5th September

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
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