Team:Cambridge/LabBook/Week11
From 2010.igem.org
EmilyKnott (Talk | contribs) |
EmilyKnott (Talk | contribs) |
||
(6 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
- | <div align="center">Week 11: Monday 20th | + | <div align="center">Week 11: Monday 20th - Sunday 26th September</div> |
==Monday== | ==Monday== | ||
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 | ||
+ | *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 | ||
+ | |||
+ | {|class="wikitable" | ||
+ | |- | ||
+ | | | ||
+ | |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): | ||
+ | {|class="wikitable" | ||
+ | |- | ||
+ | | | ||
+ | |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: | ||
+ | {|class="wikitable" | ||
+ | |- | ||
+ | |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 |
Latest revision as of 12:04, 7 October 2010
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