Objectives

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

Materials

Stock concentrations

PA: ~20 ng/µL (from PCR purification 17/09)
PB: ~20 ng/µL (from PCR purification 17/09)
pSB1C3-RFP 3: 104.14 ng/µL (from mini prep 19/07)

Quantity of DNA required for the ligation of PA and PB into pSB1C3:

PA: Digestion of 75 ng (ratio 1:1 = 55.19 ng of digested PA needed)
PB: Digestion of 75 ng (ratio 2:1 = 50.10 ng of digested PB needed)
pSB1C3-RFP: Digestion of 200 ng (50 ng per part needed —> 100 ng of digested pSB1C3 needed —> 152 ng needed)

Protocol

Digestion

In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :

• Short Spin Centrifugation
• Incubation 1h at 37°C
• Store at 4°C before gel electrophoresis and purification

Electrophoresis for digested 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 5 µL of Gel Red 10,000X (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 24 µL of each samples.

Plan:

Run at 100V

Gel purification for digested pSB1C3:

QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available on this link).

1. Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.231 g
2. Add 3 volumes Buffer QG (693 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 PA and PB:

QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available on this link).

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.

1. Mix for 5 samples (Total volume of Mix: 230 µL), in an Eppendorf tube:

   • 198.75 µL H2O

   • 25 µL Buffer Taq (1 X final, NEB #B9014S)

   • 5 µL dNTP (200 µM final, NEB #N0447S)

   • 1.25 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)

2. Add in 4 PCR tubes, in the following order:

   • 46 µL Mix

   • 1 µL primer forward (A12 or BBB-F)

   • 1 µL primer reverse (BBA-R or A13)

   • 2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)

3. —> Gently mix the reaction and perform a short spin centrifugation

4. Set the following parameters for the PCR reaction :

   • PA (2285 bp)

   • Lid temperature: 95°C

   • Initial denaturation : 95°C, 30s

   • 30 cycles of :

     • 95°C, 30 s

     • 58°C, 60 s

     • 68°C, 2 min 17 s

   • Final extension : 68°C, 5 min

   • Hold : 4°C

   • PB (1037 bp)

   • Lid temperature: 95°C

   • Initial denaturation : 95°C, 30s

   • 30 cycles of :

     • 95°C, 30 s

     • 58°C, 60 s

     • 68°C, 1 min 02 s

   • Final extension : 68°C, 5 min

   • Hold : 4°C

Electrophoresis: for screening the PCR results

1% Agarose gel:

1. Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL

2. Mix and heat it 2 min 30 s in the microwave. 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 2 µL of Purple loading dye 6 X in 10 µL of sample

3. Drop-off 10 µL of Purple ladder and 12 µL of each samples.



4. Run at 90 V.

PCR Purification

QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available on this link).

1. Add 5 volumes Buffer PB (250 µL) to 1 volume of the PCR reaction (50 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 -20°C.

Results

Electrophoresis

Expected results / Obtained results