October 9th 2016

PCR colony: on colonies transformed with BBG1

NB: BBG1 is the ligation product of BB1 (Pr-RBS) and G2 (Gluc-term)

Objectives

The overall purpose is to check if the bacteria obtain from the transformations with BBG1 contain the good genetic constructions.

Materials

Bacteria tansformed with BBG1 (made on 08/10/16).
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

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

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 7 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 :

G1 (1164 bp)
Lid temperature: 95°C
Initial denaturation : 95°C, 5 min
30 cycles of :
- 95°C, 30 s
- 58°C, 60 s
- 68°C, 1 min 13 s
Final extension : 68°C, 5 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:

Run at 90 V.

Mini-culture: bacteria transformed with BBG1

4 Mini-cultures of bacteria transformed with BBG1 (2, 4, 5, 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

Expected results / Obtained results:

Interpretation

We obtain the desired strip for BBG1 for all the colonies (n°1 to 7). As shown on the gel above, the strips are closed to 1,164 bp, which is the size of G1. A sequencing is necessary to be sure of the obtained biobrick.