NB: BB123 is the ligation product of P3 + BB12 (P1+P2), therefore it is the complete biosensor.

Objectives

The overall purpose is to check if the bacteria obtain from the transformation with BB123 contain the good genetic construction.

Materials

BBacteria tansformed with BB123 (made on 02/08/16)
Primers: A12 (forward) and A13 (reverse)

Protocol

PCR

1. Mix for 8 samples of BB123 (Total volume Mix : 400 µL) in an Eppendorf tube :

332 µL H2O

40 µL Buffer Taq (1X final, NEB #B9014S)

8 µL Primer A12 (1 µM final)

8 µL Primer A13 (1 µM final)

8 µL dNTP (200 µM final, NEB #N0447S)

4 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)

2. Add in 8 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 :

P123 (3.4 kb)
Lid température: 95°C
Initial denaturation: 95°C, 5 min
30 cycles of: 95°C, 30 s
58°C, 1 min
68°C, 3 min 24 s
Final extension: 68°C, 5 min
Hold: 4°C

Short Spin Centrifugation.

Incubation 1 h at 37°C.

Store at 4°C before gel electrophoresis or purification.

Centrifuge for 10 min at 13,000 rpm.

Electrophoresis for screening the PCR results

1% Agarose gel:

1. Put 1 g agarose + 100 mL TAE 1X in a bottle of 500 mL

2. Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid.

3. Add 5 µL of Gel Red 10,000 X (0.5 X final)

4. Flow the gel and place the combs

5. Wait until it is solidified. Remove slowly the combs.

Drop-off:

1. Short Speed centrifugation of samples.

2. Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample.

3. Drop-off 10 µL of Purple ladder and 12 µL of each samples.



4. Run at 90 V.

Miniculture of bacteria transformed with BB123

2 Mini-cultures of bacteria transformed with BB123 (6,7).
Put the colony with satisfying PCR results from the plates divided into squares to a 50 mL Falcon tube containing 5 mL LB+Cm.

Results

Electrophoresis for screening the PCR results

Expected results / Obtained results:

Interpretation

We obtain the desired strip for BB123, for the colony 6 and 7. As shown on the gel above, the strips are close to 3.4 kb, which is the size of P123. A sequencing is necessary to be sure of the obtained biobrick. The others colonies show any strips, they contain the plasmid pSB1C3 that have been close up on itself.