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Kỹ thuật Western blot

Posted date: 01:13 / comment : 0 ,

Western Blot
I. Protein extraction.
Preparation of extraction buffer for V=50mL:
Tris-HCl pH=8
final conc. 60mM
200mM=15mL
500mM=6mL
SDS
2%
10%=10mL
20%=5mL
Sucrose
15%
7.5g
miliQ water up to 50mL

Vtot=50mL
Mix well and let at RT for using to avoid precipitation (fresh use)
Add inhibitor 50X
- Grind sample in liquid N into fine powder
- Add ~ 400ul extraction buffer
- Centrifuge 15 000 rpm for 10min at 40C
- Transfer supernatant into new tube, quantify and store at -80 for later using
II. Quantification:
Prepare mixture of reagent A+B with ratio 50:1 by volume (50 vol.A+1 vol.B) in a falcon covered by aluminum foil.
- Aliquot 200ul mixture (A+B) into wells:
First row for standard: well A to I and blank
Standard
Concentration (ug/mL)
A
2000
B
1500
C
1000
D
750
E
500
F
250
G
125
H
25
I
0
Blank
0

Sample #
- Add 25ul standard conc. A to I, 25ul water for blank well
- Add 23ul miliQ + 2 ul sample (V=25ul) for each sample
- Incubate well array for 20 min at 370C
- Read single wavelength at 562nm in nanodrop
III. Preparation sample for loading and electrophoresis:
10ul of loading sample = V sample + V H2O + 0.75ul DTT0.8M 20X + 3.75ul loading dye. Protein conc. = 50ug/ul each well so prepare 15ul for each sample volume
Mixture will be pre-heated at 950C (ready at 950C) for denaturation for 5 min
Electrophoresis buffer for 500mL (1L for 2 gels): - 0.5g sodium bisulfite
                                                                                - 100mL 5x running buffer
                                                                                - dH2O up to 500mL
- Prepare the gel assembly
- Place the gel (20 wells with 10uL well) into electrophoresis machine
- Load 5uL marker proteins into first well
- Load samples # 10uL
- Load 5ul marker proteins close to the last sample well.
- Run at 175mA current or 200V for 1 gel.
IV. Prepare materials for transferring
- Whatman paper: 8 pieces in size of 12.5 x 7 cm
- PVDP membrane
- After finishing electrophoresis: transfer the gel into a tray which containing transferring buffer
- Start saturation the PVDP membrane in:
(1) Methanol and shaking: 2 min
(2) Water: 2 min
(3) Transfer buffer: 10 min
- Wet the surface of the transfer tank with transfer buffer (~100mL) and place the wet membrane on
- Cleaning the gel and cut wells off
- Wet the surface of machine
- Put 4 wet whatman papers (wet by transfer buffer) (12.5x7cm) on surface then
- Membrane with a cut at corner for orientation
- Put the gel on the membrane then
- 4 wet whatman papers
*Note: avoid bubbles at these steps
- Wipe out the extra buffer outside the sandwich with tissue
- Cover the lid and run for 70 min at 97.5mA, V 200, W 100
- Saturate the membrane by ~100mL buffer (1 TTBS 10X buffer: 10 milk v:v)
- Shaking for 30 min
- Washing for 10 min in 1X TBBS buffer
1st antibody solution: Shaking and incubating for 2h
- Decant the 1st antibody (re-use ~3 times)
- Wash with washing buffer for 3 min
- Transfer the membrane into a new tray containing washing buffer and shaking for 15 min
- Wash 3 times more with washing buffer 5 min for each time.
2nd antibody solution: Shaking and incubating for 1h
- Wash with TBBS buffer as done for 1st antibody solution.
- The membrane can be remained in TBBS buffer unstill the detection process is ready or store in 1X TBS at 40C (the membrane should be immersed completely)
- Detection: ratio 1:1 luminol enhancer: peroxide buffer (Vtot~3ml for each membrane)
- Place the membrane on a tray (flat) and drop detection solution evenly on the surface of the membrane, covering the entire surface
- Cover the membrane by 2 plastic sheets, avoid bubbles
- Visualize in image document system:
- The door open: on chip integration
                        Second 0.2~0.3
                        Preview………capture
                        Close the door…….save
Dynamic……..80 images……..interval  20s
Lights off
Capture …..auto…..save the best one

- Take the image at the best force and condition
Close the door……

- Set up parameters for image docmument 

Ly trích RNA dùng cho phân tích qPCR

Posted date: 01:12 / comment : 0 ,

RNA EXTRACTION
 SỬ DỤNG ATA BUFFER
1. Grind samples in liquid N
2. Add 600 ul extraction buffer
            5-10 ul mercaptoethanil
            2 ul antifoam
3. Transfer suspension to clean eppendorf
4. Add 84ul KCl 3M and keep on ice 15’
5. Centrifuge 8000rpm for 5’ at 40C
6. Transfer supernatant to new tube
7. Add an equal volume of LiCl2 8M (finally get LiCl2 4M)
8. Keep at 40C over night
----
9. Centrifuge 15000 rpm 20’ at 40C
10. Discard supernatant; add 400 ul DEPC H2O to pellet
11. Vortex, add 40 ul NaOAC 3M then vortex
12. Add 450 ul phenol:chloroform (1:1) then vortex
13. Centrifuge at 15000 rpm for 5’ at room temp.
14. Transfer the upper aqueous phase into new tube
15. Add an equal volume of chloroform
16. Mix and centrifuge at 15000 rpm for 5’ at 40C
17. Transfer the upper aqueous phase into new tube
18. Add 2.5 volume of cold ethanol absolute
19. Add 10 ul NaOAC
20. Keep -200C for 30’
21. Centrifuge 15000 rpm 10’ at 40C
22. Discard supernatant and air dry
23. Dissolve the pellet in 50 ul DEPC H2O
---------
Check RNA quality by electrophoresis
V= 5uL DEPC H2O + 2uL formaldehyde dye + 2uL RNA

Loading to the gel 1%



SỬ DỤNG KIT
Spectrum Plant Total RNA kit
Note: all reagents are already to use!
1. Lyse Tissue Sample
- Grind tissue sample (100mg) into fine powder
- Add 500ul of the Lysis Solution/2-ME Mixture to powder and vortex and vigorously for at least 30s
- Incubate sample at 560C for 3-5 min
2. Pellet Cellular Debris: Centrifuge the sample at max speed for 3 min
3. Filter lysate
- Pipette the lysate supernatant into a Filtration column (blue retainer ring) seated in a 2-ml collection tube. Avoid taking the pellet.
- Close the cap and centrifuge at max speed for 1 min to remove residual debris
- Save the clarified flow-through lysate!!!
4. Bind RNA to column
- Add 250ul Binding Solution to the clarified lysate and mix immediately and thoroughly by pipetting at least 5 times. Do not centrifuge.
- Bind RNA: pipette the mixture into Binding Column (red retainer ring) seated in a 2-ml collection tube. Close the cap and centrifuge at max speed for 1 min.
- Decant flow-through liquid and tap the collection tube (upside down) briefly on clean tissue paper to drain residual liquid.
- Return the column to the collection tube.
5. Column Washes:
1st wash
- Add 500ul Wash Solution 1 into the column. Close cap and centrifuge at max speed for 1 min
- Decant the flow-through and tap collection tube (upside down) briefly on clean tissue paper then return column to collection tube
2nd wash
- Add 500ul diluted Wash Solution 2 into column. Close cap and centrifuge at max speed for 30s.
- Discard the flow-through liquid and tap collection tube (upside down) on clean tissue paper for draining residual liquid. Return column to collection tube.
3rd wash
- Add 500ul diluted Wash Solution 2 into column. Close tap and centrifuge at max speed for 30s.
- Discard the flow-through and tap collection tube (upside down) on clean tissue paper for draining residual liquid. Return column to collection tube.
6. Dry Column
- Centrifuge column at max speed for 1 min to dry.
- Carefully remove the column-tube assembly from the centrifuge to avoid splashing the residual flow-through liquid on the dried column. If the residual flow-through does accidentally contact the column, re-centrifuge for 30s.
7. Elution RNA
1st elution
- Transfer the column to a new, clean 2-ml collection tube
- Add 50ul Elution Solution directly onto the center of the binding matrix.
- Close cap and let the tube sit for 1 min.
- Centrifuge at max speed for 1 min to elute.
Note: purified RNA is now in the flow-through eluate and ready for immediate use or store at -200C (short term) or – 800C (long term)
2nd elution (optional).
If the expected RNA yield is >20mg, an additional 10-30% of RNA yield may be recovered from the column with the 2nd elution.
- Transfer the column to a new, clean 2-ml collection tube.
- Add 50 ul Elution Solution directly onto center of the binding matrix
- Centrifuge at max speed for 1 min to elute.
 Note: purified RNA is now in the flow-through eluate and ready for immediate use or store at -200C (short term) or – 800C (long term)

Genotyping

Posted date: 09:32 / comment : 0 ,


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