Team:Ionis Paris/05 07 16

PCR: on P3

Objectives

The overall purpose is to amplify our geneBlock DNA fragment (P3) for further digestions and ligations in order to construct biobricks.

Materials

DNA fragments (gBlocks®):
  • P3: Part 3, 1215 bp (Term-Pu-RBS-GLuc-Term), synthesised by IDT. Primers: A12 (forward) and A13 (reverse)

  • Protocol

    PCR:
    1. Mix for 3 samples of P3 (Total volume of Mix : 144 µL), in an Eppendorf tube:

      • 119.25 µL H2O

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

      • 3 µL Primer A12 (1 µM final)

      • 3 µL Primer A13 (1 µM final)

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

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

    2. Add in 2 PCR tubes, in the respected order:

      • 50 µL Mix

      • 2 µL DNA (P1) or 2 µL H2O (control)

    3. Gently mix the reaction

    4. Short spin centrifugation

    5. Set the following parameters for the PCR reaction :

      • P1 (532 bp)

      • Lid temperature: 95°C

      • Initial denaturation: 95°C, 30 s

      • 35 cycles as such:

        • 95°C, 30 s

        • 58°C, 1 min

        • 68°C, 68°C 1min 12s

      • Final extension: 68°C, 5 min

      • Hold: 4°C

    Electrophoresis (PCR results screening)

    On a 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 (Purple loading at 1X final).
      NB : The rest of the samples (40µL) was kept for PCR purification

    3. Drop-off 10 µL of Purple ladder and 12 µL of each samples.

    4. Run at 90V

    Gel purification of P3:

    QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier(available here)

    1. Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.289 g

    2. Add 3 volumes Buffer QG (867 µL) to 1 volume of gel.

    3. Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel. The color of the mixture is yellow.

    4. Add 1 gel volume isopropanol to the sample and mix.

    5. Load 800 µL of each samples to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Load the rest and spin again.

    6. Add 500 µL Buffer QG. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

    7. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

    8. Centrifuge once more for 1 min at 13,000 rpm.

    9. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.

    10. Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

    11. Store the purified DNA at 4°C before the ligation.

    PCR purification of P3:

    QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier(available here)

    1. Add 5 volumes Buffer PB (250 µL) to 1 volume of the PCR reaction (50 µL) and mix. The color of the mixture is yellow.

    2. Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

    3. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

    4. Centrifuge once more for 1 min at 13,000 rpm.

    5. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.

    6. Add 50 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

    7. Calculate the quantity of DNA with the Nanodrop.

    8. Store the purified DNA at -20°C.

    Results

    Electrophoresis

    Expected results / Obtained results:

    We obtained expected results.

    NanoDrop Quantification

    NB: In the case of PCR purification, the ratio 260/280 were close to 1.8 meaning that our samples are quite pure.

    Interpretation

    It seems that the part 3 have been properly amplified. This stock will be kept at -20°C and used for the biosensor construction.

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