Team:Cambridge/LabBook/Week11
From 2010.igem.org
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 |
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 7.0 | 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 |
- Arabinose: 100µM
- Luciferin: 100µM
- 3 wells with Arabinose only, and 3 blank wells. Buffer at 7.
- 100µl per well
- 50µl of cells
- 48.5µl of Buffer
- 1.5µl of Luc/1µl of caged Luc
- 1µl of Arabinose
111. Expt: continuation of Biobrick assembly of pBAD with P.P.Luc(1), P.P.Luc(2), L.C.Luc(1), P.P.pSB1C3 (Emily)
- Ligation: Accidentally forgot to restrict P.P.pSB1C3 so will do that later. Followed ligation protocol on p91 with following quantities:
P.P.Luc(1) | P.P.Luc(2) | |
Vector DNA (µl) | 7.8 | 8 |
pBAD (3:1 excess) (µl) | 7.2 | 1 |
5x Rapid Ligation Buffer (µl) | 4 | 4 |
T4 DNA Ligase (µl) | 1 | 1 |
Nuclease-free H20 (µl) | 0 | 4.6 |
Cells that were ligated were then transformed
There was not enough pBAD to ligate L.C.Luc(1) ==> PCR lots of pBAD.
pBAD Colony PCR
Mix in 3x PCR tubes:
Volume (µl) | |
Nuclease-free H20 | 20 |
2x Phusion MasterMix | 25 |
VF2 Primer | 2.5 |
VR Primer | 2.5 |
pBAD stab | stab from colony |
50 |
Run program from p101.
- PCR Purification: using Qiagen kit
- Nanodrop:
- Tube 1 --> 36.7ng/µl
- Tube 2 --> 69.1ng/µl
- Tube 3 --> 66.2ng/µl
- Restriction: Following protocol on p88 with these quantities:
pBAD (1) | PP Luc (2) | PP pSB1C3 | pBAD (2) | pBAD (3) | |
Nuclease-free H20 | 0 | 14 | 13 | 1.6 | 1 |
10x FD Buffer | 2 | 2 | 2 | 2 | 2 |
Plasmid DNA | 16 | 2 | 3 | 14.4 | 15 |
EcoRI | 1 | 1 | 1 | 1 | 1 |
SpeI | 1 | 0 | 0 | 1 | 1 |
XbaI | 0 | 1 | 1 | 0 | 0 |
- Ran on a gel with 17µl DNA + 3µl of 6x orange LD in this order: Hyperladder I, pBAD 1, pBAD 2, pBAD 3, PP Luc2, PP pSB1C3
- All bands were as expected: pBAD - 1200bp, PP Luc(2) - 3600bp, PP pSB1C3 - ~4500bp
- Ben gel extracted
Ligation
Following protocol on p91, incubating at RT for 30 mins. The nanodrop reading for PPLuc(2) was so bad (2.8ng/µl) that the ligation was not performed because PPLuc(1) had already been transformed with a small amount of pBAD yesterday. Wait for results of that.
Nanodrops:
- pBAD 1 -> 3.2ng/µl
- pBAD 2 -> 34.5ng/µl
- pBAD 3 -> 15.3ng/µl
- PP pSB1C3 -> 11.5ng/µl
- LC Luc(1) -> 53.2ng/µl
PP pSB1C3 | LC Luc(1) | |
Vector DNA (µl) | 11.9 | 5.9 |
pBAD 2 (3:1 excess) (µl) | 3.1 | 9.1 |
5x Rapid Ligation Buffer (µl) | 4 | 4 |
T4 DNA Ligase (µl) | 1 | 1 |
Nuclease-free H20 | 0 | 0 |
Cells were then transformed.
Results
- Glowing growth on PPLuc(2) plates and PP pSB1C3 plates
- Growth but no glow on LC Luc(1) plates
- No growth on PP Luc(1) plates
Results later that day:
- LC Luc(1) now glowing :)
Thursday
112. Expt: Plate reader experiment. G28 and effect of D-luciferin on LC+LRE
This experiment was designed to measure the effect of Arabinose on G28 light output and D-luc level on LC/Red mutant. The layout was as follows (Blanks contain 30µl H20 + 70µl LB):
0µM | 1µM | 3µM | 10µM | |
B | ooo | ooo | ooo | ooo |
30µM | 100µM | 300µM | 1mM | |
C | ooo | ooo | ooo | ooo |
3mM | 10mM | Blank | ||
D | ooo | ooo | ooo | |
0µM | 1µM | 3µM | 10µM | |
E | ooo | ooo | ooo | ooo |
30µM | 100µM | 300µM | 1mM | |
F | ooo | ooo | ooo | ooo |
Cells, no luc, no Ara | Blank 16:H20, 84:LB | |||
G | ooo | ooo |
- B-D: Arabinose level varied in G28
- E-G: D-luc level varied in LC (red)
- 3 times repeats were made of the concentration given above. Total cell volume was 70µl for G28, 84µl for LC (red) and a total of 100µl per well.
Friday
113. Expt: Cultures for Mexico (Peter)
Grew up liquid culture of:
- 1. G28
- 2. PP Luc + LRE
- 3. pBAD + PP Luc + LRE
- 4. LC Luc + LRE
- 5. pBAD + LC Luc + LRE
Saturday
Plasmid miniprepped all 5 cultures. Nanodrop results:
1. | 269.5ng/µl |
2. | 142.1ng/µl |
3. | 187.8ng/µl |
4. | 203.1ng/µl |
5. | 176.4ng/µl |
in 50µl of EB.
Placed cryo tubes in freezer