Team:Cambridge/LabBook/Week9

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Week 9: Monday 6th September - Sunday 12th September

Contents

Monday

86. Expt: Extract CDABEG from pJS555

  • Using 2 primers
    • prefix start of O
    • suffix end of G
  • Hope that it will glow in its own right without luxR+I
  • Need to put under a new promoter

87. Expt: Gibson transformation (cont. from p.70)

3 colonies on one plate were not pink. These were streaked out on new Chl plated and put into liquid cultures along with 2 of the red colonies.

Next check for the right sized fragment with colony PCR and try to induce with arabinose.

88. Expt: Plate Reader G28

Well Arabinose conc.
A1 0 x1,9
A2 1µM
A3 5µM
A4 10µM
A5 100µM
A6 Blank
A7 pSB1C3
A8 Blank x8,16

Reads every 10 mins

89. Expt: Extracting YFP, CFP, Luc tetR, G28 via miniprep (Paul)

Nanodrop readings (ng/µl)
YFP 33.3, 55.4
CFP 17.9, 20.4
Luc tetR 33.7
G28 125.3, 96.9
G28? 17.9

90. Expt: Colony PCR to extract the thioesterase gene from E. coli K12 (Paul)

We used TOP10 cells, a substrain of DH10B, which is a substrain of K12.

We did a colony PCR to isolate the gene:

3 replicates with: 1 negative control:
2x Phusion Mastermix 10µl 10µl
Template DNA 1µl 0
Primer 1 1µl 1µl
Primer 2 1µl 1µl
Nuclease-free H20 7µl 8µl

The negative control was to check for primer/dimer and contamination.

PCR protocol:

  • Initial denaturation: 5min @ 98°C
  • Touchdown: 16 cycles
    • Denaturation: 10s @ 98°C
    • Annealing: 20s @ 65°C to 57°C
    • Elongation: 15s @ 72°C
  • 30 cycles
    • Denaturation: 10s @ 98°C
    • Annealing: 20s @ 50°C
    • Elongation: 15s @ 72°C
  • End and hold at 10°C
  • We then ran result on a gel with SyberSafe at 6x lodaing Dye and Hyperladder IV


Lane: 1 2 3 4 5
Ladder Tube 1 Tube 2 Tube 3 Negative Control

Failed: Something obtained in control...

Nothing in other lanes

We repeated without touchdown PCR:

  • Denaturation 5min @ 98°C
  • 35 Cycles
    • Denaturation: 12s @ 98°C
    • Annealing: 20s @ 58.5°C
    • Elongation: 15s @ 72°C
  • Hold at 10°C

Failed again: Product in negative control observed again...


Tuesday

91. Expt: Further testing

Column Arabiniose/µl Well names
Repeat 1 Repeat 2 Repeat 3
1 0 X1 X12 X23
2 0 X2 X13 X24
3 0 X3 X14 X25
4 5 X4 X15 X26
5 10 X5 X16 X27
6 50 X6 X17 X28
7 100 X7 X18 X29
8 1000 X8 X19 X30
9 10,000 X9 X20 X31
10 Blank X10 X21 X32
11 Blank X11 X22 X33

Inoculation: 1ml overnight added to 3ml, Amp + Cm

92. Expt: Restriction Enzyme Digests

DNA Enzymes used
1. Linear Plasmid EP
2. Luc TetR ES
3. Luc TetR EP
4. RBS YFP XP
5. RBS CFP XP
6. G28 lux SP

=

µl°C