Team:Cambridge/LabBook/Week11
From 2010.igem.org
(Difference between revisions)
EmilyKnott (Talk | contribs) |
EmilyKnott (Talk | contribs) |
||
Line 144: | Line 144: | ||
|3 | |3 | ||
|} | |} | ||
- | |||
===110. Expt: Plate reading experiment to investigate effect of pH on light output=== | ===110. Expt: Plate reading experiment to investigate effect of pH on light output=== | ||
Line 158: | Line 157: | ||
|pH 5.3 | |pH 5.3 | ||
|pH 6.1 | |pH 6.1 | ||
- | |pH 7 | + | |pH 7.0 |
|No buffer | |No buffer | ||
|- | |- | ||
Line 173: | Line 172: | ||
|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: | ||
+ | {|class="wikitable" | ||
+ | |- | ||
+ | | | ||
+ | |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: | ||
+ | {|class="wikitable" | ||
+ | | | ||
+ | |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: | ||
+ | {|class="wikitable" | ||
+ | |- | ||
+ | | | ||
+ | |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 | ||
+ | µl°C |
Revision as of 11:47, 6 October 2010
Week 11: Monday 20th September - Sunday 26th September
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
µl°C