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) :



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

2. Short Spin Centrifugation

3. Incubation 1h at 37°C

4. 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.

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 :



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

2. Mix by pipetting

3. Short spin centrifugation

4. Ligation O/N at 16°C