Team:Greece United/Experiments

In order to implement our design, we had to perform certain experiments. Our project consisted of 2 basic experimental parts. The Genetic construct assembly and the transfection of HEK293T in order to measure the effects that our construct caused. We only managed to perform the experiments for the 1st part of our experimental design. You can find all of the protocols we used in our project below:

Protocols
IDT gene Fragment suspension (1000ng)	Materials - IDT Gene Fragment - Water for Injection Equipment - Incubator at 50 ℃ - Mini centrifuge - Vortex mixer Protocol - Spin down the IDT gene Fragment tube before opening it in the mini centrifuge. - Add 100μl of water for injection - Vortex briefly. - Incubate at 50 ℃ for 15-20 min. - Vortex briefly. - Spin down the minicentrifuge.
Double digestions	Materials - DNA to be digested - Enzymes to be used - A common buffer for the 2 enzymes - Water for Injection Equipment - Microcentrifuge tubes - Incubator at 37 ℃ Protocol - Add the 1μg of DNA in a microcentrifuge tube. - Add 5μl of buffer. - Add 1μl of each enzyme (10units). - Add water for injection to a final volume of 50μl. - Incubate at 37 ℃. The incubation time highly depends on the enzyme.
Gel extraction	Materials - Agarose gel with DNA that is desired to be exctracted - QIAquick Extraction Kit - Buffer GQ - Isopropanol - 3M sodium Acetate Equipment - QIAquick spin column - Laboratory blade - Blue Light source - Scale - 2ml Microcentrifuge tube - Incubator at 50 ℃ Protocol - Place the agarose gel over a blue light source - Excise the gel slice containing the desired DNA band with a clean laboratory blade. - Place the gel slice in a microcentrifuge tube. - Tare the scale with an empty microcentrifuge tube. - Weigh the gel slice. Maximum 400mg for 1 QIAquick column. - Add 3 volumes of buffer GQ to 1 volume of 1% agarose gel. (300μl for 100mg of gel). For 2% agarose gel add 6 volumes of buffer GQ. - Incubate the tube at 50 ℃ for approximately 10 min (until gel is fully dissolved. Vortex every 2-3 min to help the gel dissolve. - Check the color of the mixture, it should be yellow. - If it is not Yellow (orange or violet), add 10 μl of 3M Sodium Acetate and mix. - Add 1 volume of isopropanol and mix (100μl for 100mg of 1%gel) - Place a QIAquick spin column in a 2 ml collection tube. - Add the dissolved gel to the QIAquick spin column. - Centrifuge for 1 min at 17,900 x g (13,000 rpm) and discard the flow through. - Add 500μl of buffer GQ to the column and centrifuge for 1 min at 17,900 x g (13,000 rpm). Discard the flow-through. - Add 750μl of buffer PE into the column, let the buffer in the column for 5 min and then centrifuge for 1 min at 17,900 x g (13,000 rpm). Discard the flow-through. - Centrifuge for 1 min at 17,900 x g (13,000 rpm) to remove residual buffer PE. - Place the column in a microcentrifuge tube. - Add 30μl of Elution Buffer (buffer EB) to the center of the column. Let stand for 4 min. Centrifuge for 1 min at 17,900 x g (13,000 rpm). - The DNA from the agarose gel slice that was cut is purified.
SpeedVac Vacuum Concentration	Materials - Sample for condensation Equipment - SpeedVac Vacuum Concentrator - Microcentrifuge tube Protocol - Measure the volume of the sample. - Load the same volume of water in another microcentrifuge tube. - Place the tubes on the concentrator antidiametrically. - Select centrifugation time depending on the volume of the sample and the desired final volume and start the centrifugation. - Measure the volume of your sample and if it is not the desired repeat the process.
Ligation	Materials - Digested plasmid DNA - Digested insert DNA - T4 DNA ligase - T4 DNA ligase buffer Equipment - Microcentrifuge tube Protocol - Add a maximum of 18 μl DNA. The plasmid to insert ratio is usually 1:3 so the amount of DNA depends on the plasmid and insert size. - Add 2μl of T4 DNA Ligase buffer. - Add 1μl of T4 DNA ligase. - If needed, add water for injection for a final reaction volume of 20μl. - Let the reaction proceed overnight at RT (25 ℃).
10 LB agar+Amp(100μg/ml) plates	Materials - Pre-mixed LB-agar powder - Ampicillin stock solution (50mg/ml) - ddH₂O Equipment - Autoclave - Durant flask (500ml) - Heat resistant gloves - Microwave oven - 10 Petri dishes (60 mm x 15 mm) - Ice bucket - Scale - Sterilization tape - Working flame Protocol - Fill the Duran flask with 120ml of dd H₂O - Measure 4.4 g of premixed LB-agar powder (37g for 1L) - Dissolve the LB-agar powder in the Duran Flask - Heat the flask without the lid in the microwave oven to fully dissolve the LB-agar powder. Avoid boiling the solution. Use a heat resistant glove for this step - Place the lid on the flask. Leave it half open. - Place a sterilization tape on top of the lid - Autoclave the solution in the autoclave for 30 min at 37 ℃, 20psi with the lid half open. The sterilization tape should darken by the end. - Spray and wipe the laboratory bench in which the plate pouring will be held with 70% ethanol. Label 10 petri dishes and stack them on your bench. - Let the dissolved LB-agar temperature drop to 60℃. To speed up the process place the side of the flask under running water and mix gently. Do not let any of the water touch the neck or top of the flask as this water is not likely to be sterile. Avoid letting the solution for too long as the LB-agar might solidify. - Place the ampicillin stock(50mg/ml) in your ice bucket. - Light a working flame in the place the LB-agar will be poured. - Add 240μl of Ampicillin(50mg/ml) in the flask. Close the flask and mix gently to spread ampicillin evenly. - Pour the solution in one plate, enough to cover 80% of the surface of the dish. Close the dish and swirl the plate to cover all of the surface. - Repeat for the remaining plates. - Leave the plates on the bench to solidify and dry overnight on the bench.
LB broth	Materials - LB broth powder - ddH₂O Equipment - Autoclave - Durant flask (500ml) - Heat resistant gloves - Microwave oven - Scale - Sterilization tape Protocol - Fill the Duran flask with 200ml of ddH₂O - Measure 4 g of premixed LB-agar powder (20g for 1L) - Dissolve the LB-broth powder in the Duran Flask - Heat the flask without the lid in the microwave oven to fully dissolve the LB-agar powder. Avoid boiling the solution. Use a heat resistant glove for this step - Place the lid on the flask. Leave it half open. - Place a sterilization tape on top of the lid. - Autoclave the solution in the autoclave for 30 min at 37℃, 20psi with the lid half open. The sterilization tape should darken by the end. - Let the LB-broth cool down and fully close the lid. - Any antibiotic can be added at this stage.
DH5 alpha transformation with Ampicillin resistance plasmid	Materials - Competent DH5a bacteria - DNA - LB-agar+Amp(100μg/ml) - LB broth Equipment - Shaking incubator at 37 °C - Stationary incubator at 37 °C - Water bath at 42 °C - Ice bucket filled with ice - Microcentrifuge tubes - Sterile spreading device - Pasteur Pipettes - Working Flame Protocol - Spray and wipe the laboratory bench with 70% ethanol. - Thaw the competent cells (taken from -80) on ice for approximately 30 min. - Place LB-agar+Amp plates at 37℃ or at RT (25℃). - Light a working flame on the bench. - Mix 1-10 DNA (1-100ng) into 20-100 μl of competent cells in a microcentrifuge tube. Mix gently. - Let the mixture on ice for 30 min. - Place 1/3 to 2/3 of the microcentrifuge tube in the water bath (42℃) for 45 sec. - Place the tube back on ice for 2 min. - Add 1ml of LB broth and incubate in a shaking incubator for 30-45 min at 37℃. - Pour 50μl of transformed cells on a LB-agar+Amp(100μg/ml) plate. - Hold the tip of a Pasteur Pipette on the working flame for ~5 sec until it moltens to seal it. Molten the Pasteur Pipette 2-3cm from the tip until it bends ~90°. - Streak the bacteria on the plate with the Pasteur pipette. - Centrifuge the rest of the transformation mix for 1-2 min at ~8.000 rpm to pellet the rest of the bacteria. - Resuspend the bacteria by pipetting. - Pour the resuspended on another LB-agar+Amp(100μg/μl) plate and repeat the procedure. - Incubate the plates overnight at 37℃ with the plates upside down (the lid on the down side).
Bacteria Inoculation in LB-broth+Amp(100μg/ml)	Materials - LB broth - Ampicillin stock (50mg/ml) - LB-agar+Amp(100μg/ml) cultures Equipment - 5ml Falcon tubes - Sterile tips - Shaking incubator at 37℃ - Pipette filler - Sterile serological pipettes - Ice bucket filled with ice Protocol - Spray and wipe the laboratory bench with 70% ethanol. - Light a working flame on the bench. - Label the falcon tubes that are going to be used. - Fill the falcon tubes with 5 ml of LB broth. - Add 10μl of Ampicillin stock(50mg/ml) in each falcon tube. - Pick a single colony from the LB-agar+Amp(100μg/ml) culture using a sterile tip and let the tip inside a falcon tube containing LB broth+Amp(100μg/ml). - Close the falcons with the lid Half way through and stabilize the lid using tape. - Incubate the liquid cultures overnight at 37℃ at 200 rpm.
QIAprep Spin Miniprep Plasmid DNA Purification	Materials - QIAspin Miniprep kit - Buffer P1 - Buffer P2 - Buffer N3 - Buffer PE - Elution Buffer (Buffer EB) - Liquid Culture of bacteria in LB-broth Equipment - QIAprep 2.0 spin column - Centrifuge - Microcentrifuge tips Protocol - Centrifuge the bacterial cultures at 8.9rpm (6800 g) for 3 min at room temperature. - Discard the supernatant. - Resuspend the pellet with 250μl of buffer P1. Vortex if needed. Transfer each bacteria culture in a microcentrifuge tube. - Add 250μl of buffer P2 and mix by inverting the tubes 4-6 times. Do not allow for the lysis reaction to proceed for more than 5 min. - Add 350μl of buffer N3 and mix immediately by inverting the tube 4-6 times. - Centrifuge for 10min at 13.000rpm(~17,900 g). - Transfer 800μl of the supernatant in a QIAprep 2.0 spin column. - Centrifuge for 1 min at 13.000rpm(~17,900 g) and discard the flow through. - Add 750μl of buffer PE. Centrifuge for 1 min at 13.000rpm(~17,900 g) and discard the flow through. - Centrifuge for 1 min at 13.000rpm(~17,900 g) to remove residual wash buffer. - Place the QIAprep 2.0 column in a clean microcentrifuge tube. - Add 50μl of Elution Buffer (Buffer EB) to the center of the QIAprep 2.0 column, let stand for 1 min and centrifuge for 1 min at 13.000rpm(~17,900 g). - The isolated purified DNA is collected in the microcentrifuge tube diluted in Elution Buffer to a final volume of 50μl.
Nanodrop 2000 DNA concentration measurement	Materials - ddH₂O - DNA sample - Solution that DNA sample is diluted (blank sample) Equipment - NanoDrop 2000 Spectrophotometer - Paper towel - Laboratory wipe Protocol - Open the Nanodrop 2000 softeware on the connected PC. Select “DNA” and then enter the wanted units in which the concentration is desired to be measured. - Lift the arm of the spectrophotometer and pipette 2μl of ddH₂O onto the bottom pedestal. - Lower the arm. Lift the arm and gently touch the pedestals with the laboratory wipe. - Lift the arm and pipette 1 μl of the blank sample on the bottom pedestal. - Lower the arm and click on “Blank” on the computer. - Lift the arm and gently touch both pedestals with the laboratory wipe. - Pipette 1 μl of DNA sample on the bottom pedestal and lower the arm. - Click on “Measure”. - Save the archive. - Lift the arm. Gently touch the laboratory wipe on both pedestals. - Pipette 2 μl of ddH₂O on the bottom pedestal and lower the arm. - Lift the arm and gently touch the laboratory wipe on both pedestals.
1% agarose gel electrophoresis (50ml)	Materials - Agarose basic powder - TBE 1x - Ethidium Bromide stock (10mg/ml) - 6x Loading Dye - DNA ladder - DNA sample Equipment - Casting Tray - Well combs - Microwave oven - Gel box - Voltage source - Heat resistant gloves - 100ml flask - scale Protocol - Prepare the casting trey and place the well combs. - Pour 50ml of TBE in a 100ml flask. - Measure 0.5g of agarose basic. - Dissolve 0.5g of agarose basic in the flask. - Using heat resistant gloves dissolve the agarose on the TBE by heating it in the microwave oven. Avoid boiling the solution. - Cool the solution by placing the flask under running water and. swirling gently. Do not cool the solution too much because the gel will solidify. - Add 2μl of Ethidium Bromide in the solution and mix gently. - Pour the solution in the casting trey and leave in RT(25℃) to solidify for approximately 30 min. - Remove carefully the well combs. - Place the gel in the gel box with its wells on the side of the negative pole of the gel box. - Fill the gel box with TBE until all of the gel is covered. - Dilute 5 μl of loading Dye with 25μl of DNA Sample in a microcentrifuge tube. - Carefully Load the DNA samples and Ladder in the wells of the gel. - Connect the gel box with the Voltage source. - Set the Voltage to 80-120 volts. - Observe the dye on the gel. - When the dye has nearly reached the edge of the gel, you can take the gel out of the gel box and observe it under UV light.