Team:Ionis Paris/05 07 16
The overall purpose is to amplify our geneBlock DNA fragment (P3) for further digestions and ligations in order to construct biobricks. P3: Part 3, 1215 bp (Term-Pu-RBS-GLuc-Term), synthesised by IDT.
Primers: A12 (forward) and A13 (reverse) Mix for 3 samples of P3 (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, 68°C 1min 12s 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 2 µL of Purple loading dye 6 X in 10 µL of sample (Purple loading at 1X final). Drop-off 10 µL of Purple ladder and 12 µL of each samples. Run at 90V QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier(available here) Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.289 g Add 3 volumes Buffer QG (867 µL) to 1 volume of gel. Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel. The color of the mixture is yellow. Add 1 gel volume isopropanol to the sample and mix. Load 800 µL of each samples to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Load the rest and spin again. Add 500 µL Buffer QG. 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 QIAquick column into 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. Store the purified DNA at 4°C before the ligation. 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: In the case of PCR purification, the ratio 260/280 were close to 1.8 meaning that our samples are quite pure. It seems that the part 3 have been properly amplified. This stock will be kept at -20°C and used for the biosensor construction.
PCR: on P3
Objectives
Materials
DNA fragments (gBlocks®):
Protocol
PCR:
Electrophoresis (PCR results screening)
NB : The rest of the samples (40µL) was kept for PCR purification
Gel purification of P3:
PCR purification of P3:
Results
Electrophoresis
NanoDrop Quantification
Interpretation
