Exeter/24 July 2013

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Contents

Results of liquid cultures

Some of our liquid cultures didn't work; the cells died in the antibiotic. Mildly frustrating. The failed cultures were:

  • OmpR (BBa_K098011)
  • Lambda inverter system (BBa_Q04510)

Minipreps using the Qiagen kit

We miniprepped:

  • CcaR
  • Promoter, RBS x 3 replicates
  • CcaS
  • B0034
  • Magenta
  • OmpR promoter
  • OmpR
  • YF1
  • Lambda inverta
  • Fix J
  • Yellow
  • Fix J promoter


Glycerol stocks

We made glycerol stocks of what is above, then stored them at -80oC

For future reference, to get glycerol stocks back as plates, take a loop and streak on gel. Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.


We then made a gel for digests using ethidium bromide.


NanoDrop from miniprep

Part Concentration (ng/ul)
CcaR 324.6
CcaS 264.4
Yellow 64.3
Magenta 102.7
RBS 48.9
Promoter, RBS, #1 21.5
Promoter, RBS, #2 22.6
Promoter, RBS, #3 22.0
Fix L 31.9
Fix J 35.4
YF1 27.1
OmpF 28.2


No SureClean was needed.

Digestion

We then cut the genes out of the plasmid using Xbal and Pstl. Prepare to run on a gel.

Each eppendorf contains:

  • 12ul purite water
  • 2ul 10X FastDigest Buffer w.Green
  • 0.5ul Xbal
  • 0.5ul Pstl
  • 5ul DNA

In gel

Lane Content
1 Ladder (1kb Gene Ruler)
2 Promoter, RBS, #1
3 B0034
4 CcaS
5 Fix J promoter (a.k.a Fix L)
6 Yellow
7 Promoter, RBS, #2
8 Fix J
9 OmpR promoter (a.k.a OmpF)
10 Promoter, RBS, #3
11 Magenta
12 CcaR
13 YF1
14 Ladder (1kb Gene Ruler)


We also ran a seperate gel of:

  • cph8 #3
  • cph8 #4
  • cph8 #5
  • cph8 #6
  • cph8 #7

Cut with Xbal and Pstl.

The lanes were run in the order above, flanked by a 1kb Gene ruler.