June 16th 2016

PCR: on P1

The overall purpose is to amplify our geneBlock DNA fragment (P1) for further digestions and ligations in order to construct biobricks.

DNA fragments (gBlocks®):

P1: Part 1, 532 bp (Pr - Elowitz RBS), synthesised by IDT. Primers: A12 (forward) and A13 (reverse).

Mix for 3 samples of P1 (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)
Add in 2 PCR tubes, in the respected order:
- 50 µL Mix
- 2 µL DNA (P1) or 2 µL H2O (control)
Gently mix the reaction
Short spin centrifugation
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

On a 1% Agarose gel:

Put 1 g agarose + 100 mL TAE 1X in a bottle of 500 mL
Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid.
Add 5 µL of Gel Red 10,000 X (0.5 X final)
Flow the gel and place the combs
Wait until it is solidified. Remove slowly the combs.

Drop-off:

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

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.
Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.
Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.
Centrifuge once more for 1 min at 13,000 rpm.
Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
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 .
Calculate the quantity of DNA with the Nanodrop.
Store the purified DNA at -20°C.