Team:Cambridge/LabBook/Week11

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Week 11: Monday 20th September - Sunday 26th September

Contents

Monday

Tuesday

107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)

  • Plasmids already extracted
  • Restriction digest following protocol on p88 using these quantities (in µl):
pBAD PP Luc (1) PP Luc (2) LC Luc (1)
Nuclease-free H20 14 14 14 14
10x FD Buffer 2 2 2 2
Plasmid DNA 2 2 2 2
FD EcoRI 1 1 1 1
FD SpeI 1 0 0 0
FD XbaI 0 1 1 1
  • Gel electrophoresis
    • pBAD failed - band at ~5000bp, should be 1200
    • others look about right ~ between 3 & 5kb (should be 3600bp)
  • Gel extraction - results in -20°C freezer

Wednesday

108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)

  • Miniprep EPIC pBAD and nanodrop
  • Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off

109. Expt: Biobrick assembly of pBAD + PP Luc (1), PP Luc (2), LC Luc (1), PP+pSB1C3

  • Peter 'miniprepped' PP+pSB1C3 to extract plasmid
  • pBAD was amplified after trying to grow overnight culture failed. The following protocol was used:

Add to a PCR tube:

10µl 2x Phusion Master Mix
1µl VF2 Primer
1µl VR Primer
8µl HPLC H20
20µl
  • Use stab to extract colony from pBAD plate and swirl in tube. Run following program on PCR machine saved as 'pBAD Amplification':
    • Heated lid at 110°C
    • Denaturation for 1m30s at 98°C
    • Cycle 35 times
      • Denaturation for 30s at 98°C
      • Elongation for 2m at 72°C
    • Elongation for 7m30s at 72°C
  • PCR Purification of pBAD from PCR reaction was performed using Qiagen kit
  • Restriction: repeating restriction of LC Luc (1) because tube from yesterday was dropped. Use protocol on p88 with following quantities (in µl):
pBAD LC Luc (1)
HPLC H20 0 14
10x FD Buffer 2 2
Plasmid DNA 16 (PCR product)* 2
FD EcoRI 1 1
FD XbaI 0 1
FD SpeI 1 0

'*' Nanodrop said 29ng/µl after PCR Purification, which was strangely low. So used 16µl as recommended (in order to not exceed 0.2µg) but maximise amount of DNA.

  • Gel electrophoresis: Run gel with following quantities:
pBAD LC Luc (1)
DNA 17 17
6x Orange LD 3 3

µl°C

110. Expt: Plate reading experiment to investigate effect of pH on light output

3pH: 5.3, 6.1, 7

D-luciferin and Caged D-luciferin (at 10mM mDMSO)

Plate layout:

pH 5.3 pH 6.1 pH 7 No buffer
D-luc o o o o o o o o o o o o
Caged D-luc o o o o o o o o o o o o