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 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 - Ligation reaction: Incubate the reaction mixture in a PCR amplifier at 50℃ for 30 min then at 4℃ preparing for transformation.
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 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℃.
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再来例子 Market Analysis
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