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Friday 27^th September

Gel SPCR7-10, NEB liquid culture, Ligation, Electrocompetent cells and transformation

Gel SPCR7-10
1%, 100V, 1h

Picture: 13/09/27_SPCR7_8_9_10 - PCRs worked


NEB liquid culture
1:1000 of ON culture

Ligation

pS006

Reagent	Volume
SPCR5	1 µl
SPCR7	0,28 µl
H2O	7.22 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS006’

Reagent	Volume
SPCR5	1 µl
lin pSB1C3 (XbaI/SpeI)	0,47 µl
H2O	7.03 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS006*

Reagent	Volume
SPCR5	1 µl
lin pSB1A3 (XbaI/SpeI)	0,67 µl
H2O	6.83 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS013

Reagent	Volume
SPCR4	1 µl
SPCR11	0,24 µl
H2O	7.26 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS013'

Reagent	Volume
lin pSB1C3 (EcoRI, SpeI)	1 µl
SPCR11	0,28 µl
H2O	7.22 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS013*

Reagent	Volume
lin pSB1A3 (EcoRI, PstI))	1 µl
SPCR11	0,3 µl
H2O	7.2 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS004

Reagent	Volume
SPCR4	1 µl
SPCR3	0,2 µl
H2O	7.3 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS004'

Reagent	Volume
lin pSB1C3 (EcoRI, PstI)	1 µl
SPCR3	0,3 µl
H2O	7.2 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS004*

Reagent	Volume
lin pSB1A3 (EcoRI, PstI)	1 µl
SPCR3	0,27 µl
H2O	7.23 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS002'

Reagent	Volume
SPCR2	1 µl
lin pSB1C3 (EcoRI, PstI)	0,8 µl
H2O	6.7 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

pS002*

Reagent	Volume
SPCR2	1 µl
lin pSB1A3 (EcoRI, PstI)	0,6 µl
H2O	6.9 µl
Fermentas T4 Ligase Buffer	1 µl
Fermentas T4 Ligase Enzime	0.5 µl
Total Volume	10 µl

45min at 22°, 15 min at 16°

Electrocompetent cells
1) Start Culture
2) When the cells reach an OD600 of 0.2.
3) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8 (OD: 0,683)
Centrifuge at 4 °C for 10 min at 4000 rpm.
4) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 25 ml of ice cold water
5) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
6) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 ul of washed cells per transformation.

Transformation
pS006, pS006’, pS006*, pS013, pS013’, pS013*, pS004, pS004’, pS004*, pS002’, pS002*

1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 1/2 h at 37 °C with shaking.

9) Plate 10/200 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.