Team:Ionis Paris/26 07 16

July 26th 2016

Digestion: P2 and pSB1C3-RFP

Objectives

Double digestion of P2 and pSB1C3-RFP by EcoRI and PstI, for the subsequent ligation of P2 in pSB1C3.

Materials

Stock concentrations:

P2: 16.82 ng/µL (from PCR purification 05/07)

pSB1C3-RFP 3: 104.14 ng/µL (from mini prep 19/07)

Quantity of DNA required for the subsequent ligation:

P2 : Digestion of 80 ng (ratio 1:1 = 41.06 ng ; ratio 2:1 = 20.53 ng —> 61,59 ng of digested P2 needed)

pSB1C3-RFP: Digestion of 190 ng (25 ng needed per ratio—> 75 ng needed)

Protocol

Digestion:

1. In a 1.5mL 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 1h at 37°C

4. Store at 4°C before gel electrophoresis and purification

Electrophoresis for pSB1C3-RFP:

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.

Loading:

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 24 µL of sample.

Plan:



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 = 0.268 g

2. Add 3 volumes Buffer QG (804 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 P2:

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 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.

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.

Digestion: P3 and pSB1C3-RFP

Objectives

Double digestion of P3 and pSB1C3-RFP by EcoRI and PstI, for the subsequent ligation of P3 in pSB1C3.

Materials

Stock concentrations:

P3: 24.64 ng/µL (from PCR purification 05/07)
pSB1C3-RFP 2: 95.204 ng/µL (from mini prep 19/07)

Quantity of DNA required for the subsequent ligation:

P3: Digestion of 100 ng (ratio 1:1 = 15 ng needed, ratio 2:1 = 30 ng needed —> 45 ng of digested P3 needed)
pSB1C3-RFP: Digestion of 100 ng (25 ng needed per ratio—> 75 ng 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 1h at 37°C

4. Store at 4°C before gel electrophoresis and purification

Electrophoresis for pSB1C3-RFP:

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.

Loading:

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 24 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 = 0.324 g

2. Add 3 volumes Buffer QG (972 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.

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.

Ligation of P2 and P3 in pSB1C3 to obtain BB2 and BB3 :

Objectives

Ligation of P2 and P3 into pSB1C3 in order to obtain 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: 3.3 ng/µL (100 ng / 30 µL)
P2: 1.6 ng/µL (80 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 fragment 2 1:1 and in 30 µL for the fragment 2 2:1 and 3 1:1, and in 40µl for the fragment 3 2:1

2. Mix by pipetting and incubate O/N at 16°C.

Mini prep: on DH5⍺ transformed with BB1

Objectives

Purification and quantification of the BB1 plasmid extracted from bacterial mini-cultures in order to get our first biobricks and use it to construct the whole Biosensor.

Materials

11 Mini-cultures of bacteria transformed with BB1 realised the 25/07 (put a colony with satisfying PCR results in 5 mL LB+CHL into a 50 mL Falcon tube).
From those mini-cultures, take 500 µL to realize a glycerol stock of tranformed bacteria. The 4.5 mL remaining will serve for the miniprep.

Protocol

The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier (available here)

Miniprep:

1. Divide each 4.5 mL bacterial O/N mini-cultures into 4 Eppendorf tubes and centrifuge all those tubes at 9,000 rpm for 3 min at room temperature. Discard the supernatant.

2. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube.

3. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue.

4. AAdd 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.

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

6. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through.

7. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

8. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

9. Centrifuge once more for 1 min at 13,000 rpm.

10. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube.

11. Add 50 μL Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

12. Calculate the quantity of DNA with the Nanodrop.

13. Store the purified DNA at -20°C.

Bacteria storage :

1. Add 100 µL of glycerol to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.
   22 tubes of BB1(2 per mini-cultures)

2. Store at -80°C.

Results

Nanodrop quantification:





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