NB: BB2, BB3 and BB12 are the ligation products of P2+ pSB1C3, P3 + pSB1C3 and BB1 + P2.

Objectives

The overall purpose is to check if the bacteria obtain from the transformation with BB2, BB3 and BB12 contain the good genetic construction.

Materials

Bacteria tansformed with BB2, BB3 and BB12(made on 27/07/16)
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

1. Mix for 17 samples for BB12 (Total volume Mix : 850 µL) in a Eppendorf tube :

705.5 µL H2O

17 µl Primer A12 (1 µM final)

17 µL Primer A13 (1 µM final)

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

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

2. Mix for 17 samples for BB2 (Total volume Mix : 850 µL) in a Eppendorf tube :

705.5 µL H2O

17 µl Primer A12 (1 µM final)

17 µL Primer A13 (1 µM final)

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

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

3. Mix for 17 samples for BB3 (Total volume Mix : 850 µL) in a Eppendorf tube :

705.5 µL H2O

17 µl Primer A12 (1 µM final)

17 µL Primer A13 (1 µM final)

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

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

4. Add in 48 PCR tubes, in the respected order:

50 µL Mix

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

5. Short spin centrifugation

6. Set the following parameters for the PCR reaction :

P12 (2.2 kb)
Lid température 95°C
Initial denaturation : 95°C, 5 min
30 cycles of : 95°C, 30 s
58°C, 1 min
68°C, 2 min 12 s
Final extension : 68°C, 5 min
Hold : 4°C/

P2 (1.7 kb)
Lid température 95°C
Initial denaturation : 95°C, 5 min
30 cycles of : 95°C, 30 s
58°C, 1 min
68°C, 1 min 42 s
Final extension : 68°C, 5 min
Hold : 4°C/

P3 (1.2 kb)
Lid température 95°C
Initial denaturation : 95°C, 5 min
30 cycles of : 95°C, 30 s
58°C, 1 min
68°C, 1 min 12 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.



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

NB: The tube with colonie 38 was damage during the PCR.

Miniculture of bacteria transformed with BB12, BB2 and BB3

6 Mini-cultures of bacteria transformed with BB12 (1, 4, 5, 7, 8, 9)
8 Mini-cultures of bacteria transformed with BB12 (33, 35, 36, 37, 39, 40, 42, 43)
1 Mini-cultures of bacteria transformed with BB3 (27)
Put the colony with satisfying PCR results from the new divided into squares petri dish to a 50mL Falcon tube containing 5mL LB+CHL.

Results

Electrophoresis:

1st electrophoresis: Expected results / Obtained results:

2nd electrophoresis: Expected results / Obtained results:

3rd electrophoresis: Expected results / Obtained results:

4th electrophoresis: Expected results / Obtained results:

Interpretation

We obtain the desired strip for BB12, for the colony 1, 4, 5, 7, 8, 9, 10, 12, 13, 14 and 15. As shown on the gel above, the strips are closed to 2.2 kb, which is the size of P1+P2. A sequencing is necessary to be sure of the obtained biobrick. The others strips are closed to 500 bp, which is the size of P1 alone.

We obtain the desired strip for BB3 for the colony 27. As shown on the gel above, the strips are close to 1.2 kb, which is the size of P3. 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.

We obtain the desired strip for BB2 for the colony 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 45, 46. As shown on the gel above, the strips are close to 1.7 kb, which is the size of P2. The colony 44 shows any strips, it contains the plasmid pSB1C3 that have been close up on itself.