Team:Worldshaper-Wuhan/procedure
Your Custom Text Here 1. Run PCR
Material required: - dnTP - DNA Primer - taq enzyme - water - template gene - buffer solution 2.
Gel electrophoresis
i. Add 3 gram of agarose ii. Add 30 ml of TAE electrophoresis buffer
iii. Stir the solution and put into microwave until the solution boils, repeat 3 times
iv. Cool down the solution to 60 Celsius degree, add gold view and stir the solution.
v. Put the solution into the cell, put in the comb according to the length of the strand.
vi. Leave it still for 15-20 minutes, take out the gel after it cooled down.
vii. Place the glycerin-added sample and marker into the gel tank
viii. Change the voltage to 120v and run it for 20 min.
3.
Gel recycling
i. Put the finished gel under the gel imaging analyzer to observe the result
ii. Cut the florescent portion of the gel and place it into a 3ml test-tube (weighed). Add 2 times the mass of the gel solution of liquid gel.
iii. Place the tube in a 55 ℃ hot bath until it completely dissolves.
iv. Add the solution obtained in the previous step to the adsorption column and place the adsorption column into the collection tube
v. Place the collection tube in a centrifuge, centrifuge at 12,000 rpm for one minute, remove the waste from the collection waste
vi. Add 600 μl of rinse solution W2 to the adsorption column and centrifuge at 12,000 rpm for 30 seconds, discard the waste in the collection tube
vii. Add 500 μl of rinse solution W2 to the adsorption column, centrifuge for 30 seconds at 12000 rpm, and discard the waste from the collection tube
viii. Place the adsorption column in a collection tube and centrifuge at 12000 rpm for 2 minutes to remove the rinse as much as possible. Place the adsorption column at room temperature for 2 minutes to dry
ix. Place the column in a clean centrifuge tube and incubate 40 μl of dd water into the middle of the adsorbent membrane for 2 minutes at room temperature. Centrifuge at 12000 rpm for 1 minute and repeat step 9 once to collect the DNA solution.
4.
Enzyme digestion
i. Raw materials: three enzymes, deoxyribonucleic acid fragments, water, buffer, plasmid
ii. inject enzyme, buffer and water into the tube containing the fragments and plasmids.
iii. The tube was put into a 37 ° C water bath and digested for 4 hours.
iv. the tube from the water bath and recycle the glue. v. Store the recovered DNA fragment at 4 ° C. vi. Add another enzyme to the tube containing the plasmid.
vii. Put the tube into a 37 ° C water bath and digest for 4 hours.
viii. Remove the tube from the water bath, recycle the gel and put it at 4℃.
5.
Ligation
- PFU enzyme and H2O
i. Add cleaved DNA fragments and cleaved plasmid fragments into test tube.
ii. Add PFU enzyme and water. Place it into PCR machine and run at 16 ° C for 1 hour.
6.
Measure concentration
i. Rinse the trace quartz cuvette with 100 μl of dd water.
ii. Repeat step 1
iii. Add 99 μl of water to the cuvette and add 1 μl of the linked plasmid sample.
iv. Place the cuvette in the spectrophotometer to read the concentration. Multiply the reading by 5000
v. Remove the plasmid and clean the cuvette with dd water
vi. Repeat steps 3 through 5 until the desired sample concentration is measured
7.
Making a medium
i. Mix 1 gram of Tryptone, 1 gram of NaCl, 0.5 g of yeast and add water to 100 ml.
ii. Mix the solution with a magnetic stirrer.
iii. Distribute the stirred solution into different test tubes and sterilize them with autoclave for 30 min
iv. Remove the solution, store some under 4 ° C and add others with agarose powder and store at 4 ° C after it solidify
8.
Transformation (shake bacteria, coated plate, pick bacteria, elution)
i. Shake bacteria - Take the medium from 4 ° C
- Add the plasmid into the competent cell. Ice for 30 min, heat for 1.5 min and - Ice again for 2 min or more
- Place the test tube on a shaker at 37 ° C and 150 rpm for 1 hour
ii. Coat plate - Remove the solid medium from 4 ° C. - Take the competent bacteria from the shaker - Swab the competent bacteria on the medium - Immerse sticks into alcohol twice
- After the stick cooled down, use it to spread the bacteria by drawing ‘z’ shape on the medium.
- After that, seal the medium, mark the corresponding date and bacteria, culture the medium in the incubator overnight at 37 ° C (up to 12 hours).
iii. Pick the bacteria - Remove the cultured dish from incubator
- Use the tip of the gun to spot the bacteria to remove the bacteria
- Throw the tip that has the bacteria into liquid medium and put it into the shake at 150 rpm overnight.
- Extract the cultured bacteria from the cultured medium
iv. Elution
- Add 500 μl of equilibration solution BL to the adsorption column CP3, centrifuge for 1 minute at 12000 rpm, discard the waste from the collection tube, and return the adsorption column to the collection tube.
- Take 1-5 ml of bacterial solution, add to the centrifuge tube, centrifuge 1 minute at 12000 rpm, try to remove the supernatant.
- Add 250 μl of solution P1 to the centrifuge tube where the bacteria are left to precipitate, use the gun to stir until no precipitate can be observed by naked eye
- Add 250 μl of P2 to the centrifuge tube, gently flip the tube 6-8 times. So that the cell lysis.
- Add 350 μl to the centrifuge tube P3, immediately gently flip the tube up and down 6-8 times until fully mixed, this time there will be white flocculent precipitation.
- Transfer the supernatant collected from the previous step to the suction column P3 and place it in the collection tube (do not draw the precipitate).
- Use 12000rpm to centrifuge 30-60 seconds, drained the waste in the collection tube, place the adsorption column into the collection tube.
- Add 600 μl of rinse PW solution to the adsorption column, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste in the collection tube, and place the column into the collection tube.
- Repeat step 7
- Place the adsorption column in a clean collection tube and centrifuge at 12000 rpm for 2 minutes to remove the remaining rinse solution from the adsorption column.
- Place the adsorption column in a clean centrifuge tube, add 50-100 μl of the elution buffer to the middle of the adsorbent membrane, allow the mixture to stand for 2 minutes at room temperature, centrifuge at 12000 rpm for 2 minutes and collect the plasmid solution into the centrifuge tube.
9.
Sequencing
i. Gel electrophoresis (see instruction above)
ii. Send the plasmid for sequencing after observing the line.
1. Culture Conditions
i. Atmosphere: CO2, 5%； ii. Temperature: 37 °C.
2.
Complete Growth Medium
i. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
3. Subculturing
i. Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
ii. Remove and discard culture medium
iii. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
iv. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed .
v. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
vi. Add appropriate aliquots of the cell suspension to new culture vessels.
vii.Incubate cultures at 37°C.
4. Cryopreservation
i. Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO.
ii. Storage temperature: liquid nitrogen vapor phase
Transfection
i. Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 60~70% confluency at the time of transfection.
ii. For plasmid DNA transfection experiment, we recommend using 500ng DNA per well in a 24-well plate.
iii. Add 500ng of plasmid DNA to the 25ul transfection medium opti, and mix immediately. Incubate the transfection mixture for 5 minutes at room temperature.
iv. Add 1ul of transfection reagent to the 25ul transfection medium opti, and mix immediately. Incubate the transfection mixture for 5 minutes at room temperature.
v. Allow the transfection Reagent/DNA complex to incubate at room temperature for 15 minutes to let transfection complex form. Never keep the complex longer than 30 minutes.
vi. Add the transfection complex to the cells drop wise.
vii. Replace fresh medium after 4~6 hours and continue to culture for 24~48 hours.
1. measurement of GFP
i. After transfection, the cells are digested to the 96 well plate.
ii. You must now measure the plate in your plate reader. The machine must be setup with the standard GFP settings (filters or excitation and emission wavelengths) that you will use when measuring your cells.
iii. Make sure to record all information about your instrument (wavelengths, etc.) as these will be required in the Plate Reader Form.
iv. Recommended filters: - Excitation 485nm
- Emission 530/30 (or as close to this as possible)
2. Calculate the copy number
- 1mol=6.02×1023 （copies） - 1dolton =1g/mol
- (MW)：dsDNA= The number of base×660 dolton/bases
- ssDNA= The number of base×330 dolton/bases
- ssRNA= The number of base×340 dolton/bases
- copies/ml= (6.02×1023)×(plasmids concentrations g/ml) /(DNA length×660)
- copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)
Procedure & Measurement
Plasmid construction
cell culture
Measurement
