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 = 0.250 g
2. Add 3 volumes Buffer QG (750 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.

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.

Results

Electrophoresis

Expected results / Obtained results