2020 Fudan Protocols
（页面摘要待补充）
Cell Cryopreservation
Cryopreservation is a technique that store cells at a very low temperatures (-80℃) to reduce cell metabolic damage and enable long-term storage.
- Add cryoprotectant: Add 80% glycerin 500μL and bacterial fluid 1 000μL into Cryopreservation vials. Mix upside down gently.
- Seal the vials: Stick sellotape around the label.
- Store the vials: Store prepared vials in refrigerator at -80℃.
Cell Recovery
Bacteria preserved at -80 ℃ need to be recovery to restores cell growth.
- Prepare plate: Take out the plate with relevant resistance from 4 degrees and wait for the temperature to the room temperature. Mark at the bottom of the plate.
- Recover the cell: The -80 ° C frozen strain was removed and placed on the ice quickly. Take a ring of the upper layer of melt bacteria liquid by the sterilized inoculation ring. Coated the plate and draw a line.
- Culture the recovered cell: Incubate at 37 degrees overnight./li>
PCR
PCR is performed for amplify DNA fragments in our project.
- Set up PCR system (25 µl):
Reagent Volume ddH2O To 25 µl 2 x Phanta Max Buffer 12.5 µl dNTP 0.5 µl Phanta 0.5 µl Template
Plasmid: 0.5 µl
PCR product: 1 µl
Forward Primer 1 µl Reverse Primer 1 µl - Place the PCR tubes in a PCR amplifier.
- Set up reaction program:
Procedure Temperature Time Cycle Initialization 95℃ 30s 1 Denaturation 95℃ 15s 40 Annealing 72℃ 15s Extension 72℃ 30s/kb Final elongation 72℃ 5min 1 - Incubate: Incubate at 16℃ until the PCR product is picked up.
Colony PCR
Colony PCR is performed to determine whether we insert DNA into plasmid successfully.
- Set up colony PCR system (10 µl):
Reagent Volume ddH2O To 10 µl 10 x Taq Buffer 1 µl dNTP 0.2 µl Taq DNA Polymerase 0.2 µl Colony Template 1 µl Forward Primer 0.4 µl Reverse Primer 0.4 µl - Place the PCR tubes in a PCR amplifier.
- Set up reaction program:
Procedure Temperature Time Cycle Initialization 94℃ 5min 1 Denaturation 94℃ 30s 25 Annealing 72℃ 30s Extension 72℃ 1min/kb Final elongation 72℃ 7min 1 - Incubate: Incubate at 8℃ until the PCR product is picked up.
Agarose Gel Electrophoresis
Agarose gel electrophoresis is performed to separate and confirm whether our plasmids were constructed properly.
- Make gel solution: Add 0.7g agarose, 70ml TAE buffer into a glass bottle and heat in a micro-oven for 2 min. Cool the liquid agarose gel to lower than 60℃ and decant the liquid agarose gel into an agarose gel tank. Add 8 μl EB into the liquid agarose gel and place the electrophoresis comb.
- Load the gel: Place the solid agarose gel in an electrophoresis device. Add 10xDNA Loading Buffer in the DNA sample, mix them up gently and carefully pipette the sample into the sample loading chambers.
- Electrophoresis: Cover the lid of the electrophoresis device, set the electrophoresis time and start electrophoresis.
DNA Gel Extraction
DNA Gel Extraction is to extract desired DNA from an agarose gel after agarose gel electrophoresis.
- Cut the DNA gel: place the gel under UV light and find the DNA band of the desired nucleotide length. b) Cut the gel containing desired DNA and put it into an Eppendorf tube.
- Melt the DNA gel: Use Vazyme® DNA Gel Extraction Kit. Add 300 μl buffer GDP to the Eppendorf tube and incubate at 55℃. Spin briefly.
- Pure the DNA gel: Insert a Fast Mini Columns-G into a 2 ml Collection Tube, transfer the solution maximally of 700 μl once a time to a filtration column, centrifuge at 12,000 x g for 30 ~ 60 sec. Discard the filtrate and reuse the Collection Tube, add 600 μl of Buffer GW (with ethanol added) to the filtration column, centrifuge at 12,000 x g for 60 sec. Pure the left solution in the same way.
- Filtrate DNA: Discard the filtrate and reuse the Collection Tube, centrifuge the empty column at 12,000 x g for 2 min. Insert the column into a clean 1.5 ml Eppendorf tube and incubate at 55℃ for 5 min. Add 7 μl ~ 30 μl of ddH2O (incubated at 55℃ in advance) to the center of the column membrane, incubate at room temperature for 2 min, and then centrifuge at 12,000 x g for 1min. Discard the filtration column. Measure the DNA concentration by Nanodrop 2000.
- Store DNA at -20℃.
Restriction Enzyme Double Digestion
Double digestion reaction is performed to create mismatch ends for directional insertion.
- Add DNA fragment and vector at a mole ratio of 2:1~4:1 (total 1μg), 5 μl NEB CutSmart buffer, 1 μl of both restriction enzymes and ddH2O up to 50 μl to set up the reaction.
- Incubate at 37℃ overnight.
ClonExpress ligation reaction
ClonExpress ligation reaction is performed to insert our DNA fragments into vector.
- Set up ClonExpress ligation system:
Reagent Volume ddH2O To 5 µl DNA fragment 0.02*bp ng vector 0.01*bp ng 2 x ClonExpress Mix 2.5 µl
Plasmid transformation
Plasmid transformation is performed to transfer plasmids into the host bacteria. We transform our plasmids into DH5α E.coli to amplify them and BL21 to testify function of our plasmids.
- Thaw all reagents on ice. And add 20 μl competent E.coli cells into the ligation product.
- Heat shock at 42℃ for 45 sec and then cool the mixture on ice for 2 min.
- Add 900 μl liquid SOC or LB medium (without antibiotic) into the mixture and shaking culture at 37℃ for 1h.
- Evenly spread the liquid culture on a solid culture medium and incubate at 37℃ overnight for colonies forming on the plate.
Plasmid Miniprep (Vazyme® FastPure Plasmid Mini Kit)
Plasmid Miniprep is performed to is extract plasmid DNA from bacteria.
- Harvest 1~ 5 ml overnight cultured (12 ~ 16 hours) bacterial cells into a centrifuge tube, centrifuge at 10,000 x g for 1 min, discard the supernatant and invert the tube on the absorbent paper to dry.
- Add 250 μl of Buffer P1 (add RNase A before use), mix thoroughly by vortex or pipetting up and down.
- Add 250 μl of Buffer P2, mix thoroughly by softly inverting the tube 8 ~ 10 times to assure complete lysis.
- Add 350 μl of Buffer P3, mix gently and thoroughly by inverting the tube 8 ~ 10 times to neutralize Buffer P2 until a flocculent white precipitate forms and centrifuge at 13,000 x g for 10 min.
- Insert a FastPure DNA Mini Column into a 2 ml Collection Tube, transfer the supernatant from step 4 to the Filtration Column, centrifuge at 13,000 x g for 30 ~ 60 sec, discard the filtrate and reuse the Collection Tube.
- Add 600 μl of Buffer PW2 (with ethanol added in) to the Filtration Column, centrifuge at 13,000 x g for 30 ~ 60 sec, discard the filtrate and reuse the Collection Tube. Centrifuge the empty Filtration Column for l min at 13,000 x g.
- Insert the Column into a clean 1.5 ml microcentrifuge tube, add 30 ~ 100 μl of Elution Buffer to the center of the Column membrane, incubate at room temperature for 2 min, centrifuge at 13,000 x g for 1min. Discard the Filtration Columns, store DNA at -20℃.
SDS-PAGE
SDS-PAGE is performed for the separation of polypeptides and confirm whether our circuits expressed properly.
- Prepare 10ml 10% Running Gel solution: Add4.1 ml ddH2O, 3.3 ml 30% Acrylamide/Bis (29:1 or 37.5:1), 2.5 ml 1.5M Tris-HCl pH8.8, 100 μl 10% SDS, 50 μl 10% APS, 5 μl TEMED. Mix them thoroughly.
- Prepare 2ml 4% Stacking Gel solution: Add 1.22 ml ddH2O, 0.26ml 30% Acrylamide/Bis (29:1 or 37.5:1), 0.5ml 0.5M Tris-HCl pH6.8, 20 μl 10% SDS, 20 μl 10% APS, 2 μl TEMED. Mix them thoroughly.
- Prepare the protein sample: Add 1 ml bacterial cells per time into a centrifuge tube, centrifuge at 12,000 x g for 1 min, discard the supernatant. Dilute SDS sample buffer to 1x. Add 1 x SDS Loading to 3000 cells per 1ul, mix thoroughly by vortex or pipetting up and down until there is no visible precipitation. Incubate at 99℃ for 5 min.
- Make the gel: Assemble the gel cassette and make sure it not to leak. Fill the gel cassette with the Running Gel softly and fill up the cassette with ddH2O. Keep it still for 10~20 min until the water layer can be observed. Pour out the ddH2O completely and fill up the cassette with Stacking Gel. Insert the comb and take care not to catch bubbles under the teeth. Keep it still.
- Load the gel: Take off the cassette and assemble the gel running stand. Fill the stand with 1 x SDS running buffer and remove the combs from the gel. Mix up Marker with 1 x SDS Loading. Load 15 μl marker mixture into the wells.
- Electrophoresis: Cover the lid of the electrophoresis device, and start electrophoresis at 200 V until the dye front is nearly at the bottom of the gel.
- Stain the gel: Submerge the whole piece of the disassembled gel with 0.1% Coomassie Blue dye for 30 min.
- Destain the gel: Pour out the 0.1% Coomassie Blue dye and wash it using ddH2O. Destain with destaining solution for 30min. Change the destaining solution to destain until it clear.
- Scan the gel.
SDS-PAGE Gradient Gel
SDS-PAGE is performed for the separation of caap and confirm whether our circuits functioned properly.
- Start the device: Put the container on the electromagnetic heating device. Increase the speed of the rotor gradually to 260 RPM.
- Prepare Gel solution: 4% and 20% Gel solution, can be prepared directly according to SDS-PAGE protocol. And 1.5 times of APS concentration can be added in autumn.
- Gradient formation: Add 4% glue slowly into the valve, and then add 20% glue. Mix them by the rotor. Wait about 2 hours for the glue to concrete.
- Store the gel: Remove the solidified glue one by one. Soak them in 1×SDS, and put them into a refrigerator at 4℃ under the counter for preservation.
IPTG induction experiment
Cryopreservation is a technique that store cells at a very low temperatures (-80℃) to reduce cell metabolic damage and enable long-term storage.
- Reconnection: Take 10 ml and 5 ml LB liquid medium in two test tubes, 10ml one is used as control group and 5ml one is for experiment group. Add 500× ampicillin 20 μl and 10μl, respectively. Culture 6h. Add 1 ml bacteria solution to experiment group and divided evenly into two tubes (3mL each) for ipTG-induced and non-induced groups. Another 1ml bacteria was used to measure initial OD.
- Measure OD: Measured OD value once per 30min and once per 15min after OD reached 0.2.
- Induce: When OD value reached about 0.6, add 500mM IPTG reach final concentration of 1mM. Incubate for 3 hours.
Growth Curve Measurement
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- Seal the vials: Stick sellotape around the label.
- Store the vials: Store prepared vials in refrigerator at -80℃.
Blood Calcium Measurement
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- Seal the vials: Stick sellotape around the label.
- Store the vials: Store prepared vials in refrigerator at -80℃.
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