Team:Ionis Paris/20 07 16

Digestion : pSB1C3-RFP, P1, P2 and P3

Objectives

Double digestion of P1, P2, P3, and pSB1C3-RFP by EcoR1 and Pst1, for the subsequent ligation of our 3 parts in pSB1C3, in order to construct BB1, BB2 and BB3.

Materials

Stock concentrations:
  • pSB1C3-RFP 2 (from mini prep 19/07) —> 95.204 ng/µL

  • P1 : 18.12 ng/µL (from PCR purification 16/06)

  • P2 PCR : 16.82 ng/µL (from PCR purification 28/06)

  • P3 PCR : 24.64 ng/µL (from PCR purification 05/07)

  • Quantity of DNA required for ligation of P1, P2 and P3 into pSB1C3:

  • pSB1C3-RFP: 3 Digestion of 190 ng (25 ng needed per ratio —> 75 ng needed per part)

  • P1: Digestion of 100 ng (37.5 ng of P1 needed)

  • P2: Digestion of 150 ng (127.5 ng of P2 needed)

  • P3: Digestion of 100 ng (90 ng of P3 needed)

  • Protocol

    Digestion:
    1. In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :

    2. NB : The digestion were done in 10 µL for pBS1C3 and the P1 and P3 and 15 µL for P2.

    3. Short Spin Centrifugation

    4. Incubation 1h at 37°C

    5. Store at 4°C before gel electrophoresis and purification

    Electrophoresis for digested pSB1C3:

    1% Agarose gel:

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

    2. Mix and heat it 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid.

    3. Add 3 µL of Gel Red 10,000X (0.3X 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 6X in the 10 µL of sample.

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

    4. Run at 90 V.

    Gel purification for digested pSB1C3:

    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 = 304 g

    2. Add 3 volumes Buffer QG (612 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 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.

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

    PCR purification for digested P1, P2 and P3:

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

    1. Add 5 volumes Buffer PB (50 µL/100 µL) to 1 volume of the sample (10 µL/20 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 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.

    7. Calculate the quantity of DNA with the Nanodrop.

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

    Results

    Electrophoresis:

    Expected results / Obtained results:

    Interpretation

    The digestion of pSB1C3-RFP was efficient, we get 2 strips at the end of the electrophoresis. The strip at 2070 pb was the digested pSB1C3 that we purified for the subsequent ligation.

    Ligation : Parts 1, 2 and 3 in pSB1C3

    Objectives

    Ligation of Parts 1, 2 and 3 into pSB1C3 in order to obtain BB1, BB2 and BB3 biobricks for subsequent transformations and creation of a stock of bacteria.
    Different ligation ratios are going to be tested : 1:1, 1:2, 1:3. The molar ratios for the ligations were calculated using NEB BioCalculator (available here)

    Materials

    Concentrations of the different components after digestion and PCR purification :
  • pSB1C3: 4.2 ng/µL (125 ng / 30 µL)

  • P1: 2 ng/µL (100 ng / 50 µL)

  • P2: 3 ng/µL (150 ng / 50 µL)

  • P3: 2 ng/µL (100 ng / 50 µL)

  • Protocol

    1. In the following order, add :

    2. NB: The ligations were done in 20 µL for the part 1 and in 30 µL for the parts 2 and 3.

    3. Mix by pipetting

    4. Short spin centrifugation

    5. Ligation O/N at 16°C

    Creative and impressive building

    How you can be a super creative

    World best photographer and photography