Objectives

The overall purpose is to amplify our geneBlock DNA fragment (P2) for further digestions and ligations in order to construct biobricks.

Materials

DNA fragments (gBlocks®):

P2: Part 2, 1785 bp (XylR), synthesised by IDT. Primers: A12 (forward) and A13 (reverse).

Protocol

PCR:

1. Mix for 3 samples of P2 (Total volume of Mix : 144 µL), in an Eppendorf tube:

   • 119.25 µL H2O

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

   • 3 µL Primer A12 (1 µM final)

   • 3 µL Primer A13 (1 µM final)

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

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

2. Add in 2 PCR tubes, in the respected order:

   • 50 µL Mix

   • 2 µL DNA (P2) or 2 µL H2O (control)

3. Gently mix the reaction

4. Short spin centrifugation

5. Set the following parameters for the PCR reaction :

   • P1 (532 bp)

   • Lid temperature: 95°C

   • Initial denaturation: 95°C, 30 s

   • 35 cycles as such:

     • 95°C, 30 s

     • 58°C, 1 min

     • 68°C, 30 s

   • Final extension: 68°C, 5 min

   • Hold: 4°C

Electrophoresis (PCR results screening)

On a 1% Agarose gel:

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

2. Mix and heat it 2 min 30 s 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 2 µL of Purple loading dye 6 X in 10 µL of sample (Purple loading at 1X final).
   NB : The rest of the samples (40µL) was kept for PCR purification

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



4. Run at 90V

Gel purification of P2 :

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 = 267 g

2. Add 3 volumes Buffer QG (801 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 of P2 :

QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier(available here)

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 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 -20°C.

Results

Electrophoresis

Expected results / Obtained results

Obtained results:

We obtained expected results.

NanoDrop Quantification

NB : In the case of PCR purification, the ratio 260/280 were close to 1.8 meaning that our samples are quite pure.

Interpretation

It seems that the part 2 have been properly amplified. This stock will be kept at -20°C and used for the biosensor construction.