Team:Ionis Paris/20 07 16
Double digestion of P1, P2, P3, and pSB1C3-RFP by EcoR1 and Pst1, for the subsequent ligation of our 3 parts in pSB1C3, in order to construct BB1, BB2 and BB3. pSB1C3-RFP 2 (from mini prep 19/07) —> 95.204 ng/µL P1 : 18.12 ng/µL (from PCR purification 16/06) P2 PCR : 16.82 ng/µL (from PCR purification 28/06) P3 PCR : 24.64 ng/µL (from PCR purification 05/07) pSB1C3-RFP: 3 Digestion of 190 ng (25 ng needed per ratio —> 75 ng needed per part) P1: Digestion of 100 ng (37.5 ng of P1 needed) P2: Digestion of 150 ng (127.5 ng of P2 needed) P3: Digestion of 100 ng (90 ng of P3 needed) In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) : NB : The digestion were done in 10 µL for pBS1C3 and the P1 and P3 and 15 µL for P2. Short Spin Centrifugation Incubation 1h at 37°C Store at 4°C before gel electrophoresis and purification 1% Agarose gel: Put 1 g of agarose low melting point + 100 mL of TAE 1X in a bottle of 500 mL. Mix and heat it 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid. Add 3 µL of Gel Red 10,000X (0.3X 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 6X in the 10 µL of sample. Drop-off 10 µL of Purple ladder and 12 µL of sample. Run at 90 V. 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 = 304 g Add 3 volumes Buffer QG (612 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 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. 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 (50 µL/100 µL) to 1 volume of the sample (10 µL/20 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 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. Calculate the quantity of DNA with the Nanodrop. Store the purified DNA at 4°C before the ligation. Expected results / Obtained results: The digestion of pSB1C3-RFP was efficient, we get 2 strips at the end of the electrophoresis. The strip at 2070 pb was the digested pSB1C3 that we purified for the subsequent ligation. Ligation of Parts 1, 2 and 3 into pSB1C3 in order to obtain BB1, BB2 and BB3 biobricks for subsequent transformations and creation of a stock of bacteria. pSB1C3: 4.2 ng/µL (125 ng / 30 µL) P1: 2 ng/µL (100 ng / 50 µL) P2: 3 ng/µL (150 ng / 50 µL) P3: 2 ng/µL (100 ng / 50 µL) In the following order, add : NB: The ligations were done in 20 µL for the part 1 and in 30 µL for the parts 2 and 3. Mix by pipetting Short spin centrifugation Ligation O/N at 16°C
Digestion : pSB1C3-RFP, P1, P2 and P3
Objectives
Materials
Stock concentrations:
Quantity of DNA required for ligation of P1, P2 and P3 into pSB1C3:
Protocol
Digestion:
Electrophoresis for digested pSB1C3:
Gel purification for digested pSB1C3:
PCR purification for digested P1, P2 and P3:
Results
Electrophoresis:
Interpretation
Ligation : Parts 1, 2 and 3 in pSB1C3
Objectives
Different ligation ratios are going to be tested : 1:1, 1:2, 1:3. The molar ratios for the ligations were calculated using NEB BioCalculator (available here)Materials
Concentrations of the different components after digestion and PCR purification :
Protocol
