September 17th 2016

PCR : on C1 and C2

Objectives

The overall purpose is to amplify our 2 different DNA fragments (C1, C2) for further digestions and ligations in order to construct biobricks.

Materials

DNA fragments : C1: "Biosensor Reporter GFP", 1356 bp (Pref, Elowitz RBS, GFP gene, double terminator), synthesized by IDT.
C2: "Pr-GFP Cello", 1285 bp (Pr, Elowitz RBS, GFP gene, double terminator), synthesized by IDT.
Primers: A12 (forward) and A13 (reverse).

Protocol

PCR

Mix for 5 samples (Total volume of Mix: 240 µL), in an Eppendorf tube:
- 198.75 µL H2O
- 25 µL Buffer Taq (1 X final, NEB #B9014S)
- 5 µL Primer A12 ( 1 µM final)
- 5 µL Primer A13 (1 µM final)
- 5 µL dNTP (200 µM final, NEB #N0447S)
- 1.25 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)
Add in 4 PCR tubes, in the following order:
- 48 µL Mix
- 2 µL of DNA fragment (C1, C2) or 2 µL H20 (Controls 1, 2)
—> Gently mix the reaction and perform a short spin centrifugation
Set the following parameters for the PCR reaction :
- C1 (1356 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 22 s
- Final extension : 68°C, 5 min
- Hold : 4°C
- C2 (1314 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 17 s
- Final extension : 68°C, 5 min
- Hold : 4°C

Electrophoresis: for screening the PCR results

1% Agarose gel:

Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL
Mix and heat it 2 min 30 s in the microwave. 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:

Short Speed centrifugation of samples
Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample
Drop-off 10 µL of Purple ladder and 12 µL of each samples.

Run at 90 V.

PCR Purification

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

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 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.
Calculate the quantity of DNA with the Nanodrop.
Store the purified DNA at -20°C.