Notebook 1

Competent cells: E.Coli DH5⍺

Objectives

The objective here is to make a stock of E.Coli DH5⍺ competent cells for subsequent transformations.

Materials

Stock concentrations:

O/N DH5⍺ pre-culture (made the 21/03): O/N inoculation of 100 µL DH5⍺ into 100 mL LB.

0.1M CaCl2: prepared the 05/06

0.1M CaCl2/15% Glycerol: prepared the 05/06

Protocol

Competence:

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.