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.

Objective

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.

Objective

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.