PCR colony: on colonies transformed by BBC3, BBC45 and BBX1

NB: BBC3 is the ligation product of BBA + C45. BBC45 is the ligation product of C45 + pSB1C3-RFP. BBX1 is the ligation product of BBX5 + B0015.

Objectives

The overall purpose is to check if the bacteria obtain from the transformations with BBC3, BBX1 and BBC45 contain the good genetic constructions.

Materials

Bacteria tansformed with BBC3(made on 11/10/16).
Bacteria tansformed with BBX1 (made on 11/10/16).
Bacteria tansformed with BBC45 (made on 11/10/16).
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

1. 3 Mix for 10 samples (Total volume of each Mix : 500 µL), in an Eppendorf tube :

240 µL H2O

5 µL Primer A12 (0.5 µM final)

5 µL Primer A13 (0.5 µM final)

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

2. 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 :

• C3 (3,565 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 47 s

• Final extension : 72°C, 2 min

• Hold : 4°C

• C45 (1,280 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

• X1 (3046 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 30 s

• Final extension : 72°C, 2 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: 8 BBX1 and 4 BBC45 PCR samples.

Plan of the 2nd electrophoresis: 4 BBC45 and 9 BBC3 PCR samples.

Run at 90 V.

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

4 mini-cultures of bacteria transformed with BBX1 (3, 4, 5, 8), BBC45 (2 white colonies) and BBC3 (1, 5, 7, 8).
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.

Results

1st electrophoresis: Expected results / Obtained results:

2nd electrophoresis: Expected results / Obtained results:

Interpretation

We obtain desired strips for BBX1 and BBC3. A sequencing is necessary to be sure of the obtained biobricks.

Concerning BBC45, chosen colonies became red. These colonies integrated pSB1C3-RFP instead of BBC45. The slice of gel had to contain also pSB1C3-RFP plasmid digested at only one restriction site. We decide to choose 2 white colonies on the plates, wishing that they contain the correct plasmid BBC45. A sequencing is necessary to be sure of the obtained biobricks.