Team:Ionis Paris/16 06 16
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: Short Speed centrifugation of samples. Addition of 1 µL of Purple loading dye 6 X in 5 µL of sample. Drop-off 10 µL of Purple ladder and 6 µL of each samples. Run at 90V 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. Expected results/Obtained results: We obtained expected results. NB: The ratio 260/280 were close to 1.8 meaning that our samples are quite pure. It seems that the part 1 have been properly amplified. This stock will be kept at -20°C and used for the biosensor construction.
PCR: on P1
Objectives
Materials
Protocol
PCR:
Electrophoresis (PCR results screening)
PCR Purification
Results
Electrophoresis
NanoDrop Quantification
Interpretation
