October 11th 2016

PCR colony: on colonies transformed by BBX3, BBX5, pSB1A2-C45 and BBG3

NB: BBX3 is the ligation product of P12his + BB-B0015. BBX5 is the ligation product of BB12 + X4. pSB1A2-C45 is the ligation product of C4 + BBC5.

Objectives

The overall purpose is to check if the bacteria obtain from the transformations withBBX3, BBX5, pSB1A2-C45 and BBG3 contain the good genetic constructions.

Materials

Bacteria tansformed with BBX3 (made on 10/10/16).
Bacteria tansformed with BBX5 (made on 10/10/16).
Bacteria tansformed with pSB1A2-C45 (made on 10/10/16).
Bacteria tansformed with BBG3 (made on 10/10/16).
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

1. 1 Mix for 15 samples (Total volume of each Mix : 750µL), in an Eppendorf tube :

360 µL H2O

7.5 µL Primer A12 (0.5 µM final)

7.5 µL Primer A13 (0.5 µM final)

375 µL Q5 High-Fidelity 2X Master Mix (NEB #M0492S)

2. 1 Mix for 11 samples (Total volume of each Mix : 550µL), in an Eppendorf tube :

264 µL H2O

7.5 µL Primer A12 (1 µM final)

7.5 µL Primer A13 (1 µM final)

275 µL Q5 High-Fidelity 2X Master Mix (NEB #M0492S)

3. Add in 24 PCR tubes, in the respected order:

50 µL Mix

One colony from the different petri dishes (pick one colony, put some on a new plate, divided into squares, and put the remaining bacteria into the PCR mix)

Gently mix the reaction

3. Short spin centrifugation

4. Set the following parameters for the PCR reaction :

X3 (2312 bp)
Lid temperature: 98°C
Initial denaturation : 98°C, 5 min
30 cycles of :
- 98°C, 10 s
- 64°C, 30 s
- 72°C, 1 min 10 s
Final extension : 72°C, 2 min

Hold : 4°C

X5 (2917 bp)
Lid temperature: 98°C
Initial denaturation : 98°C, 5 min
30 cycles of :
- 98°C, 10 s
- 64°C, 30 s
- 72°C, 1 min 28 s
Final extension : 72°C, 2 min

Hold : 4°C

C45 (1280 bp)
Lid temperature: 98°C
Initial denaturation : 98°C, 5 min
30 cycles of :
- 98°C, 10 s
- 64°C, 30 s
- 72°C, 38 s
Final extension : 72°C, 2 min

Hold : 4°C

G3 (617 bp)
Lid temperature: 98°C
Initial denaturation : 98°C, 5 min
30 cycles of :
- 98°C, 10 s
- 64°C, 30 s
- 72°C, 19 s
Final extension : 72°C, 2 min

Hold : 4°C

Electrophoresis for screening the PCR results

1% Agarose gel:

Put 1 g agarose + 100 mL TAE 1X in a bottle of 500 mL
Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid.
Add 5 µL of Gel Red 10,000 X (0.5 X final)
Flow the gel and place the combs
Wait until it is solidified. Remove slowly the combs.

Drop-off:

Short Speed centrifugation of samples.
Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample.
Drop-off 10 µL of Purple ladder and 12 µL of each samples.

Plan of the 1st electrophoresis: 3 BBX5 and 7 BBX3 PCR samples.

Plan of the 2nd electrophoresis: 7 BBC45 and 7 BBG3 PCR samples.

Run at 90 V.

Mini-culture: bacteria transformed with BBX3, BBX5, pSB1A2-C45 and BBG3

4 mini-cultures of bacteria transformed with pSB1A2-C45 (4, 5, 6, 7) and 3 mini-cultures of bacteria transformed with BBX3 (1, 2, 3), BBX5 (1, 2, 3) and BBG3 (1, 2, 3).
Put the colony with satisfying PCR results from the plates divided into squares to a 50 mL Falcon tube containing 5 mL LB+Cm.

Run at 90 V.