PCR : on PA and PB

Objectives

The overall purpose is to create PA (Pr - RBS - XylR - Term) and PB (Pu - RBS - Gaussia - Term) from our biosensor.

Materials

DNA fragments : BB123mut-6 (from mini prep 07/09)
Primers: A12 (forward) and BBA-R (reverse) for PA, and BBB-F (forward) and A13 (reverse) for PB.

Protocol

PCR

1. Mix for 5 samples (Total volume of Mix: 230 µL), in an Eppendorf tube:

   • 198.75 µL H2O

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

   • 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:

   • 46 µL Mix

   • 1 µL primer forward (A12 or BBB-F)

   • 1 µL primer reverse (BBA-R or A13)

   • 2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)

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

4. Set the following parameters for the PCR reaction :

   • PA (2285 bp)

   • Lid temperature: 95°C

   • Initial denaturation : 95°C, 30s

   • 30 cycles of :

     • 95°C, 30 s

     • 58°C, 60 s

     • 68°C, 2 min 17 s

   • Final extension : 68°C, 5 min

   • Hold : 4°C

   • PB (1037 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 02 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.

PCR Purification

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

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 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.

7. Calculate the quantity of DNA with the Nanodrop.

8. Store the purified DNA at -20°C.

Results

Expected results / Obtained results: