Difference between revisions of "Team:TJU China/Experiments"

Conversion

1. Wipe the cleaned work area with 70% ethanol.
2. Thaw the competent cells on ice. A 1.5 mL microcentrifuge tube was labeled for each transformation and the tube was placed on ice for pre-cooling. .
3. Rotate the DNA tube from the Competent Cell Test Kit/Conversion Efficiency Kit and collect all DNA to the bottom of each tube before use. A quick spin of 20-30 seconds at 8,000-10,000 rpm is sufficient. Note: You should resuspend the DNA in each tube with 50 μL of dH2O.
4. Inhale 1 μL of DNA into each microcentrifuge tube.
5. Pipette 50 μL of competent cells into each tube. Gently mix the tube with your fingers.
6. Incubate on ice for 30 minutes.
Now preheat the bath to 42 °C.
7. Heat the cells by placing in a water bath for 90 seconds. Carefully keep the lid of the tube above the water level and keep the ice close.
8. Immediately transfer the tube back to the ice and incubate on ice for 5 minutes.
9. Add 950 μL of SOC medium to each tube, incubate at 37 ° C for 1 hour, and shake at 200-300 rpm.
10. Pipette 100 μL from each tube onto the appropriate plate and spread the mixture evenly over the plate. Incubate overnight at 37 ° C or for about 16 hours. Place the plate with the agar side on top and the lid on the bottom.

Kit DNA conversion

1. Using a pipette tip, make a hole in the foil cover and enter the corresponding hole in the part. Make sure the board is oriented correctly. Do not remove the foil cover as it may cause cross-contamination between the holes.
2. Pipette 10 μL of dH2O (distilled water) into the wells. Pipette up and down several times and let stand for 5 minutes to ensure that the dried DNA is completely resuspended. The resuspension will be red because the dried DNA has a cresol red dye. We recommend that you do not resuspend dry DNA with TE.
3. Convert 1 μL of resuspended DNA to the competent cells you want, transform your transformation with the appropriate antibiotic* and grow overnight.
4. Pick a colony and inoculate the broth (using the correct antibiotic again) and grow for 16 hours.
5. Using the resulting culture, make a small amount of DNA to make your own glycerol stock (further instructions on making glycerin see this page). We recommend using QC testing of micro-prepared DNA, such as restriction digestion and sequencing.

PCR

Single enzyme digestion

Take 1 ng of the verified plasmid, add BμI 1 μL, buffer 0.5 μL, add ddHO to 50 μL, place in a 37 ° C water bath for 1 h, then put in a 65 ° C water bath for 20 min, take out and run the gel verification.
connection:
First add a large fragment of 25ng, a small fragment of 1μM, 0.2μL (small fragment is pre-annealed by two complementary single-stranded oligonucleotides in a water bath in a boiling water beaker, slowly reduced to room temperature), then add T4 ligase 1μL 1 μL of its buffer, add ddHO to 20 μL, place in ice water and mix to 22 ° C in a thermos bucket, and connect overnight.
Double digestion:
Add 1 ul of enzyme, add 1 ng (or 2 ng) of plasmid, add buffer, then hydrate, 30 ul system. 37 ° C 40min (time can be changed) 80 ° C 5min fire

Extraction of the amplified plasmid from the cultured Escherichia coli using TIANprep Mini Plasmid Kit( #DP103, TIANgen)

1. Collect the E. coli solution into the EP tube.
2. Re-suspend pelleted bacterial cells in 250ul Buffer P1 (RNase A added, kept at 4 °C) and mix thoroughly.
3. Add Buffer 250ul P2 and gently invert the tube 6-8 times to mix.
4. Add 350μl Buffer P3 and invert the tube immediately and gently 6-8 times.
5. Centrifuge for 10 min at 12,000 rpm in a micro-centrifuge.
6. Regenerate column CP3 while centrifugation. Add 500μl Buffer BL. Centrifuge for 1 min at 12,000 rpm after static for 2min. Discard the flow-through.
7. Add supernatant from the EP tube to the column and put it into collection canals. Centrifuge for 1min at 12000rpm. Discard the flow-through.
8. Add 600μl Buffer PW(ethanol added) and centrifuge for 1min after static for 2min. Discard the flow-through.
9. Repeat step 8.
10. Centrifuging for 2min at 12000rpm to shake off the rest of the Buffer PW.
11. Place the column in a new EP tube and the opening was allowed to stand for 5 minutes, so that the ethanol in the PW can be sufficiently volatilized.
12. Add 70 ul sterile distilled water at 75℃ dropwise to the middle of the adsorbed film. Static for 2min. Then centrifuge for 2 min at 12,000 rpm to collect DNA solution in EP tube.

Agarose Gel Electrophoresis (Plasmid & PCR & Cleavage product)

1. Prepare sufficient lx TAE to fill the electrophoresis tank and to cast the gel.
2. Prepare a solution of agarose in electrophoresis buffer at a concentration of 1%: Add 0.9g powdered agarose to 90ml of TAE in an Erlenmeyer flask.
3. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.
4. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 ug/ml. Mix the gel solution thoroughly by gentle swirling.
5. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.
6. Pour the warm agarose solution into the mold.
7. Allow the gel to set completely (30-45 minutes at room temperature), then carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape Mount the gel in the electrophoresis tank.
8. Add just enough electrophoresis buffer to cover the gel to a depth of ~1 mm.
9. Mix the samples of DNA with 10 ul green buffer
10. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micro-pipettor. Load size standards into slots on both the right and left sides of the gel.
11. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue should migrate from the wells into the body of the gel. Run the gel until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.
12. When the DNA samples or dyes have migrated a sufficient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank.

Gel Extraction purification using TIANgen Midi Purification Kit (#DP209, TIANGEN)

1.Column equilibrium: add 500 μl equilibrium liquid BL to the adsorption column CA2 (the adsorption column into the collection tube) , centrifuge for 1 min at 12,000 rpm in a micro-centrifuge. Then pour out the waste liquid from the collection tube, and put the adsorption column back into the collection tube.
2.Cut a single DNA band from agarose gel and put it into a clean centrifuge tube to weigh.
3.Add equal volume solution PN (if gel weight is 0.1 g, add 100ul PN solution), water bath at 50 ℃, during which the EP tube is continuously flipped mildly up and down to ensure the full dissolution of the gel. If there are still undissolved rubber pieces, keep them for a few minutes or add some more sol solution until the block is completely dissolved.
4.The solution obtained from the previous step was added to column CA2 (the column was placed in the collection tube), incubate at room temperature for 2 mins, then centrifuge for 1 min at 12,000 rpm in a micro-centrifuge. Discard the flow-through and put the adsorption column CA2 into the collection tube.
5.Adding 600 μl PW(ethanol added) to the adsorption column CA2, then centrifuge for 1 min at 12,000 rpm in a micro-centrifuge. Discard the flow-through and put the adsorption column CA2 into the collection tube.
6.Repeat step 5.
7.Put the adsorption column CA2 back into the collecting tube, then centrifuge for 2 min at 12,000 rpm in a micro-centrifuge in order to remove the rinsing solution as much as possible. The adsorption column CA2 was then placed at room temperature for a few minutes and dried thoroughly to prevent the residual rinsing solution from affecting the next step.
8.Place the adsorption column CA2 in a clean EP tube, add 40-50ul double distilled water (pH 7.0-8.5) to the middle of the adsorption membrane, incubate at room temperature for 2 mins. Then centrifuge for 2 min at 12,000 rpm in a micro-centrifuge to collect DNA solution.