(Difference between revisions)
Line 362: Line 362:
Line 376: Line 376:
Line 390: Line 390:
Line 404: Line 404:

Revision as of 13:57, 1 October 2010

Week 10: Monday 13th September - Sunday 19th September



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

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

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


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)


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
FD EcoRI 1
FD SpeI 1
Nuclease-free H20 10
  • 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

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

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

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