Team:Ionis Paris/Notebook/11 09 16

PCR : on C1 and C2

Objectives

The overall purpose is to amplify our 2 different DNA fragments (C1, C2) for further digestions and ligations in order to construct biobricks.

Materials

DNA fragments : C1: "Biosensor Reporter GFP", 1356 bp (Pref, Elowitz RBS, GFP gene, double terminator), synthesized by IDT.
C2: "Pr-GFP Cello", 1285 bp (Pr, Elowitz RBS, GFP gene, double terminator), synthesized by IDT.
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

  1. Mix for 5 samples (Total volume of Mix: 240 µL), in an Eppendorf tube:

    • 198.75 µL H2O

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

    • 5 µL Primer A12 ( 1 µM final)

    • 5 µL Primer A13 (1 µM final)

    • 5 µL dNTP (200 µM final, NEB #N0447S)

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

  2. Add in 4 PCR tubes, in the following order:

    • 48 µL Mix

    • 2 µL of DNA fragment (C1, C2) or 2 µL H20 (Controls 1, 2)

  3. —> Gently mix the reaction and perform a short spin centrifugation

  4. Set the following parameters for the PCR reaction :

    • C1 (1356 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 22 s

    • Final extension : 68°C, 5 min

    • Hold : 4°C

    • C2 (1314 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 17 s

    • Final extension : 68°C, 5 min

    • Hold : 4°C

    5. Electrophoresis: for screening the PCR results

    1% Agarose gel:

    1. Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL

    2. Mix and heat it 2 min 30 s in the microwave. 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. Run at 90 V.

    Results

    Expected results / Obtained results

Interpretation

The PCR did not work and we did not observe DNA in the C1 stock. We will start again in case if it was linked to an error of manipulation, and we will also amplify an other DNA fragment, already successfully amplified, as a positive control for the PCR.

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