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

1. 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)

2. 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)

3. —> Gently mix the reaction and perform a short spin centrifugation

4. 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:

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

Expected results / Obtained results