Team:Ionis Paris/Notebook 1

Digestion: BB1 and C4

Objectives

Double digestion of BB1 by SpeI and PstI, and of BBC2 by XbaI and PstI for the subsequent ligation of C2 in BB1.

Materials

Stock concentrations:

  • BB1-2: 97,3 ng/µL (from mini prep realised the 26/07)

  • BBC5-1: ~175 ng/µL (from mini prep realized the 03/10)

    • Quantity of DNA required for the ligation of C5 into BB1:

  • BB1: Digestion of 50 ng (25 ng needed per ratio)

  • BBC5: Digestion of 175 ng (ratio 3:1 = 25 ng of digested C5 needed, ratio 2:1 = 16.5 ng of digested C5 needed —> 42 ng of C5 needed —> 142 ng of BBC5 needed)

    • Protocol

    Digestion:

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

  • 2. Short Spin Centrifugation

  • 3. Incubation 1h at 37°C

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

    • Electrophoresis for digested BBC5:

    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 4 µL of Purple loading dye 6X in the 20 µL of sample.

  • 3. Drop-off 10 µL of Purple ladder and 25 µL of sample.

    • Plan:

  • 4. Run at 90 V.

    • Gel purification for C5:

    QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available on https://www.qiagen.com/us/resources/resourcedetail?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en)

  • 1. Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.235 g

  • 2. Add 3 volumes Buffer QG (705 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 for digested BB1:

    QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available on https://www.qiagen.com/fi/resources/resourcedetail?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en)

  • 1. Add 5 volumes Buffer PB (100 µL) to 1 volume of the sample (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. 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 BBC5 was efficient, we get 2 strips at the end of the electrophoresis. The strip at 857 pb was the digested C5 that we purified for the subsequent ligation.

    Ligation of C5 into BB1 :

    Objectives

    Ligation of C5 into BB1 in order to obtain BBC2, for subsequent transformation and creation of a stock of bacteria.The molar ratios for the ligation were calculated using NEB BioCalculator (http://nebiocalculator.neb.com/#!/ligation).

    Materials

    Concentrations of the different components after digestion and PCR purification:

    • BB1: 3.24 ng/µL (97 ng / 30 µL)

    • C5: 1.67 ng/µL (50 ng / 30 µL)

  • 1. In the following order, add :

    • NB: The ligations were done in 30 µL

  • 2. Mix by pipetting

  • 3. Incubate for 1h at room temperature

    • For the construction of our plasmid, we selected parts that are RFC[10] compatible. In Figure 4 below, the map of our plasmid is represented in a simplified way, composed of the pSB1C3 backbone and the biosensor part.

    Creative and impressive building

    How you can be a super creative

    World best photographer and photography