Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 16th July.html

Phage Sensor

ASDF

Tuesday 16^th July

We prepared a colony PCR to send the KAN for sequencing, to know exaclty the sequence of the KAN.

KEIOΔMPYRF has a deletion of PYRF to be replaced by KAN
We pitched 4 singles colonies into 50µL of H2O
Boild 5 minutes at 95°C
1,5 µL of this can be used directly for PCR

PCR reaction
Keep all the regents at 4°C while preparing the mixture

Reagent	Volume
JW 182 (10 uM) JW 183 (10 uM) Template DNA Quick-load Tag 2x Master Mix Nuclease free water	0,5 µL 0,5 µL 1,5 µL 12,5 µL 10 µL
Total volume	25 µL

Thermocycle Protocol : NEB Quick-Load

	Temperature	Time
Start Cycle1 Cycle 2 Cycle 3 Finish Store	95°C 95°C 50°C 68°C 68°C 10°C	30 seconds 15 seconds 30 seconds 1 minute/kB 5 minutes Forever	melt melt anneal extend - 35 cycles extend store

Gel electrophorese

We do it to prouve that our colony PCR was successful. We expect a band at about 800 basepaires.
Our gel is 1%, therefore we used 0,5g agarose in 100µL TAE buffer.
As a ladder, we used a 1kB pluse gene ruler of fermentor.
We l the gel with 5 µL sample ans we kept the sample at 4°C.

PICTURE

We see that the band are as expected. THis is an indicator that KAN is at the right place. The sequence has then been sent for sequencing

Transformation of PAUC 18 into a NEBΔturbo claring cells
We are transforming PAUC 18 that consist in a low ORI and ampliciline resistance
We will use commercialized NEB turbo competent cells as well as freshly made chemical competant cells
We did this to check the competancy of our fresh competant cells

NOTE : EVERYTHING HAS TO BE KEPT ON ICE AND NO VORTEX

Throw competant cells on ice. Those can be prepared using the CaCl2 protocol
Place 20 µL of cells in a pre-chilled Eppendorf tube
For an intect vector, add 0,5 µL or less to the chilled cells
For a ligation product, add 2-3 µL to the chilled cells
Mix gently by flicking the tube
Chill on ice for 10 minutes - this step is optional but can improve yields when transforming a ligation product
Heat stock at 42°C for 30 seconds
Return on ice for 2 minutes
Add 200µL LB medium and recover the cells by shaking at 37°C
Another rich medium can substituate for the recovery. The recovery time varies with the antibiotic selection
Ampicillin : 15 - 30 minutes

Place out the cells on selective LB
Use glass beads to spread the cells. The volume of cells plated depends on what is being transformed

For an intact vector
High transformation efficiencies are expected. Plating out 10 µL of recoverd cells should produce many colonies.

Note : 200 µL is the maximum volume of liquid that an LB plate can absorb
Incubate at 37°C. Transformants should appear within 12 hours

Conclusion
The BL 21 DE3 strain showed IP colonies
For the new NEB turbo cells, we see no colony
The old NEB turbo cells worked fine