NB: BBA is the ligation product of pSB1C3 + PA, and BBB is the ligation product of pSB1C3 + PB.

Objectives

The overall purpose is to check if the bacteria obtained from the transformations with BBA and BBB contain the good genetic constructions.

Materials

Bacteria tansformed with BBA and BBB (made on 20/09/16).
Primers: A12 (forward) and A13 (reverse)

Protocol

PCR

1. 2 Mix for 15 samples (Total volume of each Mix : 750µL), in an Eppendorf tube :

622.5 µL H2O

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

15 µL Primer A12 (1 µM final)

15 µL Primer A13 (1 µM final)

15 µL dNTP (200 µM final, NEB #N0447S)

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

2. Add in 42 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 :

• PA (2285 bp)

• Lid temperature: 95°C

• Initial denaturation : 95°C, 30s

• 30 cycles of :

  • 95°C, 30 s

  • 58°C, 60 s

  • 68°C, 2 min 17 s

• Final extension : 68°C, 5 min

• Hold : 4°C

• PB (1037 bp)

• Lid temperature: 95°C

• Initial denaturation : 95°C, 30s

• 30 cycles of :

  • 95°C, 30 s

  • 58°C, 60 s

  • 68°C, 1 min 02 s

• Final extension : 68°C, 5 min

• Hold : 4°C

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.

Plan of the 1st electrophoresis: BBA PCR samples of colonies 1 to 14

Plan of the 2nd electrophoresis: BBB PCR samples of colonies 1 to 14

Run at 90 V.

Miniculture of bacteria transformed with BBA and BBB

28 Mini-cultures of bacteria transformed with BBA (n°1 to 14) and BBB (n°1 to 14):
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 (obtained on the 26/09)

1st Electrophoresis: Expected results / Obtained results:

2nd Electrophoresis: Expected results / Obtained results:

Interpretation

We obtain the desired strip for BBA, for all the colonies (n°1 to 14), excepted the n°13. As shown on the gel above, the strips are closed to 2,285 bp, which is the size of PA. A sequencing is necessary to be sure of the obtained biobrick.

We obtain the desired strip for BBB, for all the colonies (n°1 to 14). As shown on the gel above, the strips are closed to 1,037 bp, which is the size of PB. A sequencing is necessary to be sure of the obtained biobrick.