Team:Cambridge/Notebook/12

From 2010.igem.org

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*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.
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*We carried on with those that had worked with just pBAD.
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See continuation below.
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===114. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD===
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Arabinose concentration varying from 0µM to 10mM
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*1. 0µM: 0Ara/30µl of H20
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*2. 1µM: 1µl of Ara@100µM/29µl of H20
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*3. 3µM: 3µl of Ara@100µM/27µl of H20
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*4. 10µM: 10µl of Ara@100µM/20µl of H20
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*5. 30µM: 30µl of Ara@100µM/no H20
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*6. 100µM: 1µl of Ara@10mM/29µl of H20
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*7. 300µM: 3µl of Ara@10mM/27µl of H20
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*8. 1mM: 10µl of Ara@10mM/20µl of H20
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*9. 3mM: 30µl of Ara@10mM/no H20
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*10. 10mM: 1µl of Ara@1M/29µl of H20
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*11. No luciferin, 100µM Ara => 1µl of 10mM, 30.5 of H20
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D-luciferin at 100µM -> 15µl.
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Total volume 100µl per well. 60.5µl of overnight culture
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Plate layout:
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{|class="wikitable"
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|-
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|1-3
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|4-6
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|7-9
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|10-12
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|-
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|A
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|PP(1)
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|PP(2)
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|PP(3)
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|PP(4)
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|-
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|B
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|PP(5)
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|PP(6)
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|PP(7)
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|PP(8)
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|-
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|C
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|PP(9)
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|PP(10)
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|PP(11)
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|LC(5)
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|-
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|D
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|
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|
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|-
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|E
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|-
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|F
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|LC(1)
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|LC(6)
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|LC(3)
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|LC(4)
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|-
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|G
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|
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|
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|LC(7)
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|LC(8)
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|-
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|H
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|LC(9)
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|LC(10)
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|LC(11)
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|Blanks
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|}
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µl°C
µl°C

Revision as of 11:32, 7 October 2010

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

Retrieved from "http://2010.igem.org/Team:Cambridge/Notebook/12"