Digestion: BB3 and BB12

Objectives

Double digestion of BB1 by SpeI and PstI, and of BBC5 by XbaI and PstI for the subsequent ligation of C5 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 realised the 03/10)

Quantity of DNA required for the subsequent ligation:

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) :

   

   NB: The digestion were done in 20 µL.

2. Short Spin Centrifugation.

3. Incubation 1 h at 37°C.

4. Store at 4°C before gel electrophoresis or 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 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 4 µL of Purple loading dye 6X in the 20 µL of each samples.

3. Drop-off 10 µL of Purple ladder and 25 µL of each samples.



4. Run at 90 V.

Gel purification for digested C5:

Competence:

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 = 0.235 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 here)

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 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. 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: P3 into BB12

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

Materials

Concentrations of the different components after digesion 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 1 h at room temperature.

Transformation: competent DH5⍺ cells with ligation product BB123

Objectives

The objective is to transforme competent DH5⍺ cells with the ligation products BBC2.

Materials

2 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16).

Plasmid DNA: Ligation product BBC2.

Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

Experimental conditions realized :

We need 6 LB+Cm plates + 4 LB plates

Transformations protocol:

1. Thaw tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.

2. Add the 30 µL plasmid DNA to the cell mixture.

3. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.

4. Place on ice for 30 min. Do not mix.

5. Heat shock at exactly 42°C for 45 s. Do not mix.

6. Place on ice for 5 min. Do not mix.

7. Pipette 250 µL of room temperature SOC into the mixture.

8. Place at 37°C for 1h at 250 rpm.

9. Warm selection plates to 25°C.

10. Mix the cells thoroughly by flicking the tubes and inverting.

11. Spread the corresponding volume onto each plate.

12. Incubate all the plates O/N at 37°C.

Results (obtained the 05/10)

Expected results:

Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
A bacterial lawn on the LB petri dishes without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained results:

We obtained expected results. We obtained more colonies with the ratio 2:1.

Interpretation

The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. However, this resistance can be due to the plamid BB1. It is possible that BB1 has closed up on itself. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore to ensure that bacteria incorpore the correct plasmid BBC2.