Team:Cambridge/Notebook/12

From 2010.igem.org

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<div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
<div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
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==Monday==
===113. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)===
===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
*Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
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===115. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)===
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PCR mixes were:
 +
*0.25µl forward primer
 +
*0.25µl reverse primer
 +
*2µl template
 +
*22.5µl PCR water
 +
*25µl 2x Phusion Mastermix
 +
 +
*Template DNA was taken from colony (100µl PCR water + 1 colony)
 +
*Primers were taken directly from tubes (undiluted)
 +
 +
====PCR Protocol====
 +
{|class="wikitable"
 +
|-
 +
|
 +
|110°C
 +
|Heated lid
 +
|-
 +
|1m30
 +
|98°C
 +
|Denaturation
 +
|-
 +
|Cycle 30 times
 +
|
 +
|
 +
|-
 +
|30s
 +
|98°C
 +
|Denaturation
 +
|-
 +
|2m
 +
|72°C
 +
|Annealing & Elongation
 +
|-
 +
|End cycle
 +
|
 +
|
 +
|-
 +
|7m30
 +
|72°C
 +
|Final elongation
 +
|-
 +
|
 +
|10°C
 +
|Final hold
 +
|}
µl°C
µl°C

Revision as of 11:38, 7 October 2010

Week 11: Monday 27th September - Sunday 3rd October

Contents

Monday

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

115. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)

PCR mixes were:

  • 0.25µl forward primer
  • 0.25µl reverse primer
  • 2µl template
  • 22.5µl PCR water
  • 25µl 2x Phusion Mastermix
  • Template DNA was taken from colony (100µl PCR water + 1 colony)
  • Primers were taken directly from tubes (undiluted)

PCR Protocol

110°C Heated lid
1m30 98°C Denaturation
Cycle 30 times
30s 98°C Denaturation
2m 72°C Annealing & Elongation
End cycle
7m30 72°C Final elongation
10°C Final hold

µl°C

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