Latest revision as of 21:41, 18 September 2015

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Description

1. Cells transfection

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

RNA extraction

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 Transcription

1.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.

3.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.