Team:SCUT-China/Protocols
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Description
1. Cells transfectiob
1.Seed cells to be 40% confluent at a 35mm culture dish.
2.Dilute 10ul lentiviral vector in 1ml DMEM medium containing 10% FBS
3.Withdraw culture medium from 35mm culture dish.
4.Add vector-DMEM complex to cells
5.Incubate for 15 hours.
6.Withdraw vector-DMEM complex from culture dish.
7.Add 2ml DMEM medium containing 10% FBS to cells and incubate for 10 hours
8.Observe the cells under Inverted fluorescence microscope.
2. RT-PCR
1.Add trizol (3ml per culture dish);
2.Keep portions in centrifuge tube(1ml per centrifuge tube)
3.Homogenized by pipetting several times.
4.Incubate samples for 5 min at room temp.
5.Add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)
6.Shake for 15sec.
7.Incubate samples for 5 min at room temp.
8.Centrifuge11.5G, 15 min, 4 ℃.
9.Transfer 0.5ml aqueous phase to a new centrifuge tube.
10.Add isopropanol (1/2 volume of trizol; e.g. 0.5ml to 1ml)
11.Reverse blending.
12.Incubate samples for 10 min at room temp.
13.Centrifuge11.5G, 10 min, 4 ℃.
14.Discard the supernatant.
15.Add 70% EtOH (1 volume of trizol; e.g. 1ml to 1ml ,add & vortex briefly)
16.Centrifuge11.5G, 5 min, 4 ℃.
17.Discard the supernatant.
18.Air-dry pellet for 2-5min.
19.Add 20μlRNase free water and store in -70℃ environment.
20.Determine RNA content by UV spectrophotometry.
21.Electrophoresis of RNA.
Two-Step RT-PCR STEP1:Reverse Transcription1.Assemble the reaction on ice. Add the enzyme last.
2.Add the following components to a nuclease-free microcentrifuge tube.
3.Heat mixture to 65°C for 5 min and quick chill on ice. Collect the contents of the tube by brief centrifugation and add:
4.Mix contents of the tube gently and incubate at 37°C for 2 min.
5.Add 1 µl (200 units) of M-MLV RT,and mix by pipetting gently up and down.
6.Incubate 50 min at 37°C.
7.Inactivate the reaction by heating at 70°C for 15 min.
Elisa
1. Prepare all standards and samples be added in duplicate to the micro elisa stripplate.
2. Add standard : Set Standard wells , testing sample wells. Add standard 50 μl to standard well .
3. Add Sample : Add testing sample 10 μl then add Sample Diluent 40 μl to testing sample well(samples were 5 times diluted )Blank well doesn’t add anyting.
4. Add 100 μl of HRP-conjugate reagent to each well , cover with an adhesive strip and incubate for 60 minutes at 37°C .
5. Aspirate each well and wash by filling each well with Wash Solution (400μl ), repeating the process four times for a total of five washes. After the last wash, remove any remaining Wash Solution by decanting. Invert the plate and blot it against cleanpaper towels.
6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well.Gently mix and incubate for 15 minutes at 37 ℃ Protect from light .
7. Add 50μl Stop Solution to each well.
8. Read the Optical Density ( OD) at 450 nm using a Microplate Reader.