Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 4th September.html


Phage Sensor

Wednesday 4th September

Starting Liquid culture, PCRs, Gel, DpnI digest, Digestion of SPCR5,7, Purification of Digestion, Ligation of SPCR5,7, Gibson Assembly, Making Electrocompetent Cells, Transformation, Redo SPCR12/13 ,

Starting Liquid culture
NEB turbo, grow up to an OD of 0.6


Reagant Volume
1x (SPCR2) 11x (SPCR12) 11x (SPCR13) 8x (SPCR1) 8x (SPCR4)
Nuclease-free water 37.25 µl 447 µl 447 µl 335,25µl 335,25µl
5x Phusion HF Buffer 10 µl 120 µl 120 µl 90 µl 90 µl
10 mM dNTPs 1 µl 12 µl 12 µl 9 µl 9 µl
Forward Primer (10 uM) 0.5 µl 6 µl 6 µl 4.5 µl 4.5 µl
Reverse Primer (10 uM) 0.5 µl 6 µl 6 µl 4.5 µl 4.5 µl
Template Plasmid 0.25 µl 3 µl 3 µl 2.25 µl 2.25 µl
Phusion DNA Polymerase 0.5 µl 6 µl 6 µl 4,5 µl 4,5 µl
Total Volume 50 µl 600 µl 600 µl 450 µl 450 µl

for SPCR 12/13 gradient, 2,5 min
for SPCR3, SPCR11 51°, 45s
for SPCR 1, SPCR2, 48,8°, 2 min
for SPCR4, 40,8°, 1 min

1%, 100V, 20 min

1.small gel (SPCR1,2,3,4,11)
2. big gel (SPCR12)
3. big Gel (SPCR13)

DpnI digest
SPCR 1: add 10ul DpnI
SPCR4: add 10ul DpnI
SPCR2,3,11: add 1 ul DpnI

incubate for 1h at 37°

Digestion of SPCR5,7

Reagent Volume
Purified PCR Product 16 µl
H2O 0 µl
10x FastDigest Buffer 2 µl
XbaI 1 µl
SpeI 1 µl
Total Volume 20.0 µl

Incubate for 1h at 37°, shaking

Purification of Digestion
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

Ligation of SPCR5,7

Reagent Volume
Vector 1 µl
Insert 3 µl
H2O 4.5 µl
Fermentas T4 Ligase Buffer 1 µl
Fermentas T4 Ligase Enzyme 0.5 µl
Total Volume 10 µl

Incubate at room temperature for ~1h

Gibson Assembly

Amount per Reaction : Positive Control**

PCR Fragment(s)+ linearized vector : 2-10 μl (0.02–0.5 pmols)*

Gibson Assembly Master Mix (2X): 10 μl

Deionized H2O : XX μl

Total Volume : 20 μl

Incubate samples in a thermocycler at 50°C for 15 minutes.
Put on Ice
Insert: 2,4 ul
Vector: 0,6 ul

Making Electrocompetent Cells

1) Start a liquid culture of cells. NEB turbo
2) dilute the cells 100X.
100 ml LB.
Grow at 30°C for about 90 minutes.
3) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
5) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
6) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
7) 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.

pS005, pS006, SPCR3 (IDT plasmid) M13mp18
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 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.

Redo SPCR12/13
Reagent Volume
11x (SPCR12) 11x (SPCR13)
Nuclease-free water 447 µl 447 µl
5x Phusion HF Buffer 120 µl 120 µl
10 mM dNTPs 12 µl 12 µl
Forward Primer (10 uM) 6 µl 6 µl
Reverse Primer (10 uM) 6 µl 6 µl
Template Plasmid 3 µl 3 µl
Phusion DNA Polymerase 6 µl 6 µl
Total Volume 600 µl 600 µl

Cycling protocol as usual + gradient + topdown