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Team:Cambridge/LabBook/Week10
From 2010.igem.org
Contents |
Monday
100. Expt: Adding pBAD to P.P. pSB1C3 (Hannah and Emily)
- Took P.P. pSB1C3 out of fridge
- Extracted pBAD
- Restricted pBAD with E + S (using protocol from Fermentas below)
- Restricted P.P. pSB1C3
- Ran on gel
Here are gel columns:
| Lane 1 | Lane 2 | Lane 3 | Lane 4 | Lane 5 | Lane 6 | Lane 7 |
| EZ Ladder II | P.P.pSB1C3 | P.P.pSB1C3 | pBAD (1) | pBAD (1) | pBAD (2) | pBAD (2) |
| 5µl | 24µl | 12µl | 12µl | 12µl | 12µl | 12µl |
Results of gel: P.P.+pSB1C3 worked but pBAD could not be seen. P.P.+pSB1C3 was around 5kb as expected. Ben performed gel extraction on this.
We used pBAD and P.P.+pSB1C3 from a previous experiment (from the freezer) and performed ligation using the Fermentas protocol below, but using the following quantities:
| Volume (µl) | |
| P.P. pSB1C3 | 1 |
| pBAD | 1 |
| DI H20 | 13 |
| 5x Rapid ligation Buffer | 4 |
| T4 DNA Ligase | 1 |
| 20 |
Fermentas Restriction Digest Protocol
1. Combine the following reaction components at room temperature in the order indicated:
| Plasmid DNA | |
| Water, nuclease-free | 15µl |
| 10x FastDigest Buffer | 2µl |
| DNA | 2µl (up to 1µg) |
| FastDigest enzyme | 1µl |
| 20µl |
2. Mix gently and spin down
3. Incubate at 37°C in a heat block or water thermostat for 5min (we actually left them for 30 mins). Optional: Inactivate the enzyme by heating for 5min at 80°C.
Fermentas Ligation Protocol
1. Add the following to a microcentrifuge tube:
| Linearized vector DNA | 10-100ng |
| Insert DNA (at 3:1 Molar excess over vector) | variable |
| 5x Rapid Ligation Buffer | 4µl |
| T4 DNA Ligase, 5u/µl | 1µl |
| Water, nuclease-free | to 20µl |
2. Vortex and spin briefly to collect drops.
3. Incubate the mixture at 22°C (RT) for 5 mins (we used 30 mins)
4. Use 2-5µl of the ligation mixture for transformation
The reaction mixture can be stored at 0-4°C until used for transformation.
101. Expt: Transformation of plasmid obtained in experiment on p88
We transformed TOP10 cells with the plasmid extracted from the experiment on p88 using the standard protocol. We incubated overnight on Cm plates and obtained colonies on plates from plasmids of lanes: bottom; 2 top; 8; 5; 7; 8; 9.
We then grew overnight LB cultures for plasmid extraction.
The colonies had a slightly different aspect suggesting some possible contamination. I've therefore extracted plasmids from 3 lanes:
- Lane 1 bottom: 19.8ng/µl
- Lane 2 top: 5.6ng/µl
- Lane 5: 16.8ng/µl
These 3 have been sent to sequencing.
102. Expt: Making glycerol stock
In CC iGEM 2010 Green Box in -80°C Freezer:
1. DNA2.0 PP in TOP10 from source
2. DNA2.0 LC in TOP10 from source
3. PP + pSB1C3
4. LC + pSB1C3
5. LC + pBAD + pSB1C3
6. Thio Lane 1 bottom
7. Thio Lane 2 top
8. Thio Lane 5
9. Thio Lane 7
Tuesday
103. Expt: Mutagenesis of Luciola cruciata
Will's mutagenesis primers will all be sorted in the green box in the freezer once diluted. Ordered primers diluted to 100µM as suggested by Biolegio.
Working stocks will be made (to reduce freeze-thaw damage to the main stocks) at 10mM.
Primers in reactions should be at 0.5µM (approx) so 1µl of dilute primer stock per 20µl reaction.
Colony PCR
- Lyse cells first, take a loopful of colony and add 20µl of ddH20 (in a PCR tube)
- Heat at 98°C for 10 mins
- Cool to -80°C for ~10 mins
- Vortex for ~2mins
PCR mix:
| ddH20 | 7µl |
| Phusion Mast Mix (2x) | 10µl |
| F Primer | 1µl |
| R Primer | 1µl |
| Lysate | 1µl |
| 20µl reactions |
Used mutagenesis.f.LC and mut286.r.LC --> Labelled (1)
and mutagenesis.r.LC and mut286.f.LC (to mutate back to wild type) --> Labelled (2)
Started Gibson assembly with (1) and (2), products from colony PCR with 15µl Gibson master mix, 2.5µl (1), 2.5µl (2)
Wednesday
104. Expt: Adding pBAD to P.P. pSB1C3 (Hannah + Emily)
- Restriction: Use Fermentas protocol on p88 using the quantities below. Cut P.P. pSB1C3 used from experiment yesterday. Restriction performed on pBAD using E and S.
| Volume (µl) | |
| 10x FD Buffer | 2 |
| pBAD DNA | 6 |
| FD EcoRI | 1 |
| FD SpeI | 1 |
| Nuclease-free H20 | 10 |
| 20 |
- Gel: Showed 3 bands around 5100, 5000 and 1200, as expected. The gel was loaded as follows:
Easy Ladder II, pBAD, pBAD, pBAD, pBAD
- Ligation: After gel extraction, pBAD results from nanodrop were unreliable, but eventually gave stable readings of 8.2ng/µl
Ligation was performed using the protocol on p90 using the following quantities:
| Volume (µl) | |
| 5x Rapid Ligation Buffer | 4 |
| T4 DNA Ligase | 1 |
| P.P. pSB1C3 | 6.5 |
| pBAD | 8.5 |
Transformation failed :(
105. Expt: Luminescence measurements with plate reader to measure effect of Arabinose; luciferin and D-cysteine
We used 96-well plate and tested the effect of varying concentrations of Arabinose and luciferin with and without D-cysteine.
There repeats were made each time
| Luc | ||||
| 10µM | 100µM | 1mM | ||
| Arabinose | 10µM | 1-3 | 4-6 | 7-9 with DC |
| 10-12 | 13-15 | 16-18 without DC | ||
| 100µM | 19-21 | 22-24 | 25-27 with DC | |
| 28-30 | 31-33 | 34-36 without DC | ||
| 1mM | 37-39 | 40-42 | 43-45 with DC | |
| 46-48 | 49-51 | 52-54 without DC |
55-57: 100 luc, 0 Ara, No DC 58-60: 0 luc, 100 Ara, No DC 61-63: 100 luc, 100 Ara --> No cells
Cells were taken from 2 colonies of LC + pBAD + pSB1C3 and mixed in 10ml of LB.
75µl of LB + cells was added in each well + DI H20 and necessary Arabinose/luciferin in the right concentrations.
D-cysteine was added to ~1mM
Thursday
106. Expt: Gibson to separate luciferase and LRE in P. pyralis and L. cruciata (Bill, Emily and Hannah)
We diluted primers from Biolegio using the stated quantities of water on the product sheets. Then made a 1 in 10 dilution of this and followed the Finnzymes Phusion Master Mix protocol. The following was mixed for solutions A to P:
| Volume (µl) | |
| DI H20 | 19 |
| 2x Phusion Master Mix | 25 |
| Primer 1 | 2.5 |
| Primer 2 | 2.5 |
| Template DNA | 1 |
| 50 |
Primer 1, primer 2 and template DNA were as follows for each solution:
| Solution | To extract | Primer 1 | Primer 2 | Template DNA | |
| Uneven length outputs | A | LRE | 1 | 6 | PP |
| B | Ext | 4 | 7 | pSB1C3 | |
| C | Luc | 5 | 2 | PP | |
| D | Ext | 3 | 8 | pSB1C3 | |
| E | LRE | 1 | 10 | LC | |
| F | Ext | 11 | 4 | pSB1C3 | |
| G | Luc | 9 | 2 | LC | |
| H | Ext | 3 | 12 | pSB1C3 | |
| Even length outputs | I | LRE | f | 6 | PP |
| J | LRE | 7 | r | PP | |
| K | Luc | 5 | r | PP | |
| L | Luc | f | 8 | PP | |
| M | LRE | f | 10 | LC | |
| N | LRE | 11 | r | LC | |
| O | Luc | f | 12 | LC | |
| P | Luc | 9 | r | LC |
where f = forward mutagenesis primer, r = reverse mutagenesis primer, PP = P. pyralis, LC = L. cruciata
These solutions were run in the PCR machine using the following program:
| Temp (°C) | Duration | |
| Initial Denaturation | 98 | 30s |
| 35 cycles | ||
| Denaturation | 98 | 8s |
| Annealing | 67-75 | 25s |
| Elongation | 72 | 2m30s |
| End cycle | ||
| Final elongation | 72 | 10mins |
| Hold | 4 |
This experiment was first performed on 16th September and the diagnostic gel showed it had failed => the bands were all in the same places and wrong. This was because the lid had not been closed properly on the PCR machine; the hot plate was not touching the tube lids. Also, the hot lid had not been switched on so condensation formed at the tops of the tubes.
Friday
When repeated on Friday there was no condensation and the gel showed solutions A to H had all the correct lengths as expected. However, only K, M and P had worked for the remaining solutions.
So, Ben gel extracted solutions A to H.
Bill transformed and plated out the results over the weekend.
