September 12th 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 6 samples (Total volume of Mix: 288 µL), in an Eppendorf tube:
- 238.5 µL H2O
- 25 µL Buffer Taq (1 X final, NEB #B9014S)
- 6 µL Primer A12 ( 1 µM final)
- 6 µL Primer A13 (1 µM final)
- 6 µL dNTP (200 µM final, NEB #N0447S)
- 1.5 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)
Add in 5 PCR tubes, in the following order:
- 48 µL Mix
- 2 µL of DNA fragment (C1, C2,P2) 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
- P2 (1785 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 42 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.

Results

Expected results / Obtained results

Interpretation

The PCR of C1 and C2 did not work whereas P2 has been successfully amplified, and we did not observe DNA in the C1 stock as in the electrophoresis made the day before. We can conclude that IDT sent an empty tube for the C1 fragment and we can suppose that their has been a problem in the synthesis of the C2 fragment.