Team:Ionis Paris/Notebook/09 10 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:

    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:

    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.

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