Digestion: BB3 and BB12

Objectives

Double digestion of BB3 (pSB1C3-P3) by XbaI and PstI and double digestion of BB12 (pSB1C3-P1-P2) by SpeI and PstI, for the subsequent ligation of P3 in BB12 in order to obtain BB123, and thus our biosensor.

Materials

Stock concentrations:

BB3-27 : 213.3 ng/µL (from mini prep 29/07)

BB12-4 : 429.9 ng/µL (from mini prep 29/07)

Quantity of DNA required for the subsequent ligation:

BB3 : Digestion of 213.3 ng (ratio 2:1 = 28.57 ng ; ratio 3:1 = 42.86 ng —> 71.43 ng of digested P3 needed)

BB12 : Digestion of 429.9 ng (50 ng per ratio —> 100 ng of digested BB12 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.

5. Centrifuge for 10 min at 13,000 rpm.

Electrophoresis for digested BB3:

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 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 = 397 g

2. Add 3 volumes Buffer QG (1,191 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 BB12:

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. 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:

Nanodrop:

Obtained results:

Interpretation

The digestion was efficient, we get 2 strips at the end of the electrophoresis. The strip at 1200 pb was the digested P3 that we purified for the subsequent ligation.

Ligation: P3 into BB12

Objectives

Ligation of P3 into BB12 for subsequent transformation and amplification of BB12.
Different ligation ratios are going to be tested 2:1 and 3:1. The molar ratios for the ligation were calculated using NEB BioCalculator (available here)

Materials

Concentrations of the different components after digesion and PCR purification :

BB12: 6.518 ng/µL
P3: 2.37 ng/µL (71.1 ng, 1/3 of the inital quantity of BB3, into 30 µL)

Protocol

1. In the following order, add :

   

   NB: The ligations were done in 30 µLfor the ratio 2:1 and 3:1

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 BB123.

Materials

2 aliquots of DH5⍺ Competent cells (from the 23/07/16)

Plasmid DNA : Ligation product pSB1C3-P1-P2-P3

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 (obtain the 03/08)

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 obtain the expected results.

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 BB12. It is possible that BB12 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 BB123.