Team:NUDT CHINA/Experiments
EXPERIMENTS & PROTOCOL
I.Agarose gel(CLICK)
We often use 0.7%,1% and 1.5% agarose gel, take 0.7% agarose gel for example:
1. Weigh 0.175 g of agarose.
2. Add 25 mL of TAE 1X.
3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.
4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight, and that the “comb” is well placed.
5. When homogeneous, add 2 µL of SYBR SAFE DNA Gel Stain to the solution and mix well.
6. Pour the solution into the bed and clear all its bubbles with a tip.
7. Mix the samples with loading dye in a 10:1 ratio. Put the samples into the wells, as well as marker into the first well.
Note: For different size of gels , we have 25 ml ,50 ml and 100 ml agarose gel.
II.LB medium(CLICK)
LB medium (1L liquid)
1. 10 g tryptone
2. 10 g NaCl
3. 5 g yeast extract
4. ddH2O
In the late stage of the experiment, we use LB Broth produced by Sangon Biotech, 2.5g LB Broth for 100 ml liquid.
LB medium (solid, 1L = 50 dishes)
1. 15 g agar
2. 10 g tryptone
3. 10 g NaCl
4. 5 g yeast extract
4.ddH2O
In the late stage of the experiment, we use LB Broth produced by Sangon Biotech, 4 g LB Broth for 100 ml liquid.
For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin).
III.Plasmids extraction(CLICK)
For bacterial plasmid extraction we used Omega Plasmid Miniprep Kit according to: [PDF]Bacterial Plasmid Extraction
IV.Gel extraction(CLICK)
For bacterial gel extraction we used Omega gel extraction Kit according to: [PDF]Gel Extraction
V.Transformation(CLICK)
1. Mix 50 µL of competent cells with 1 µL DNA or 6 µL production of ligament.
2. Put competent cells (we use DH5α and BL21) according to: [PDF]DH5α &[PDF]BL21) on ice for 30 min and the above mix as well
3. Put them in water bath at 42°C for 45s, promptly transformed to ice for 2 min.
4. Add (in super clean bench) 450µL of LB medium.
5. Incubate for 60 min at 37 °C incubator shaker.
6. Centrifuge at 4000rpm for 2 min.
7. Spread 100 µL in the plate.
8. Cultivate at 37°C for 12-16 hours.
VI.PCR(CLICK)
1. 5µL Premix Taq
2. 0.4µL DNA
3. 0.4µL Primer-F
4. 0.4µL Primer-R
5. 3.8µL ddH2O
TOTAL: 10µL
Premix Taq according to: [PDF]Premix Taq
PCR reaction parameter:
1. 94°C 4min
2. 94°C 30s, 58°C 30s, 72°C 30s (1000kb correspond to 1 min) 35 cycles
3. 72°C 10 min
4. Stay at 4°C
VII.Ligament(CLICK)
1. 1µL Ligase(10×Buffer)
2. 1µL PEG
3. 0.5µL T4 DNA Ligase[PDF]
4. 2µL Insert
5. 2µL Vector
6. Add ddH2O to 10µL
VIII.Enzyme digestion(CLICK)
1. 2µL 10×Buffer
2. 1µL Enzyme 1
3. 1µL Enzyme 2
4. 4~6µL DNA
5. Add ddH2O to 10µL
Enzyme:
Spe1: [PDF]Spe1
EcoR1: [PDF]EcoR1
Xba1: [PDF]Xba1
Pst1: [PDF]Pst1
Quick Cut Spe1: [PDF]Quick Cut Spe1
Quick Cut EcoR1: [PDF]Quick Cut EcoR1
Quick Cut Xba1: [PDF]Quick Cut Xba1
Quick Cut Pst1: [PDF]Quick Cut Pst1
IX.RT(CLICK)
PrimeScriptTM RT reagent Kit with gDNA Eraser according to: [PDF]RT reagent Kit with gDNA Eraser
X.CHIP(CLICK)
Day 1
1. Harvest cells from 10-cm culture dish around 90% confluency. Aspirate culture medium with vacuum pump, wash with 1xPBS twice, remove all trace of fluid.
2. Add 10ml fresh 1xPBS and 270ul 37% formaldehyde (final concentration 1%).
3. Add 1ml 1.25M glycine solution to the culture dish, swirl gently, and place at room temperature for 5 minutes, this will stop crosslink of formaldehyde.
4. Wash with pre-chilled 1xPBS containing 1mM PMSF twice, aspirate all trace of fluid.
5. Add 1ml 1xPBS containing 1mM PMSF, harvest cells using scraper, and transfer cell suspension into a fresh 1.5ml microtube. Wash culture dish with another 1ml of 1xPBS containing 1mM PMSF, and transfer to another fresh 1.5ml microtube.
6. Centrifuge at 800-1000xg for 5 minutes at 4°C to pellet cells.
7. Remove supernatant and resuspend cell pellet using 200ul complete ChIP lysis buffer Remove supernatant and resuspend cell pellet using 200ul complete ChIP lysis buffer (containing 1mM PMSF, 1ug/ml leupeptin and 1ug/ml aprotinin), combine suspensions into a single microtube. Place on ice for 10 minutes.
8. Sonicate to shear cells. (10-20sec pulse/pause, 10 cycles, 30% power for HeLa cells).
9. Put them in ice for 10min.
10. Centrifuge at 14,000x g for 5 minutes at 4°C, transfer 300ul supernatant to a fresh 1.5ml microtube. Add 1.7ml ChIP Dilution Buffer (containing 1mM PMSF).
11. Take 20ul as INPUT, the other add 70ul Protein A+G Agrose/salmon sperm DNA. Rotate for 30min at 4°C.
12. Centrifuge at 1000x g for 1 minutes at 4°C, transfer the supernatant to a new 2ml microtube. Add primary antibody, Rotate for a whole night at 4°C.
Day 2
1. Add 60ul Protein A+G Agrose/salmon sperm DNA, Rotate for 30-60min at 4°C.
2. Centrifuge at 1000x g for 1 minutes at 4°C, remove the supernatant carefully.
3. Use following liquid to wash the precipitation, 1ml a time, rotate for 3-5min at 4°C, then Centrifuge at 1000x g for 1 minutes at 4°C, remove the supernatant carefully:
A. Low salt immune complex wash buffer (once)
B. High salt immune complex wash buffer (once)
C. LiCl immune complex wash buffer (once)
D. TE buffer (twice)
4. Add 250ul Elusion buffer, mix well and rotate for 3-5min at room temperature
5. Centrifuge at 1000x g for 1 minutes, transfer the supernatant to a new microtube, add 250ul Elusion buffer to the precipitate, mix well and rotate for 3-5min at room temperature.
6. Centrifuge at 1000x g for 1 minutes, transfer the supernatant to mix with the supernatant got in last step, add 20ul 5M NaCl, mix well and heat for 4 hours at 65°C.
7. Add 10ul 0.5M EDTA, 20ul Tris pH6.5, 1ul 20mg/ml Proteinase K solution, vortex and incubate at 45°C for 60 minutes.
8. Use kit to purify DNA
9. PCR
XI.ELISA(CLICK)
We use Plant Indole-3-acetic acid ELISA kit according to: [PDF]Plant Indole-3-acetic Acid ELISA Kit
XII.RNA extraction(CLICK)
1. Phase separation:
A. 15~45 min depending on number of samples and whether an additional chloroform wash is necessary.
B. Add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)shake for 15 sec (Eccles protocol: do not vortex)incubate 2-5 min at RTspin max. 12000g, 5-15 min, 2-8°Cif centrifugation hasn''t been sufficient the DNA-containing interphase will be cloud-like and poorly compacted If supernatant appears turbid an additional chloroform cleaning step can be inserted here.transfer aqueous upper phase into new tube.
C. Take care not to aspirate the DNA-containing white interface. This quickly happens and will lead to DNA contamination in your RNA prep.
D. TRIZOL phases after chloroform addition TOP - colourless aqueous phase (RNA) - 60% TRIZOL volume MIDDLE - interphase (DNA) BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids).
2. RNA precipitation and wash:
A. 20~40 min depending on number of samples.
B. Add isopropanol (70% of aqueous phase or 1/2 trizol volume)0.8 M sodium citrate or 1.2 M NaCl can be added(incubate 10min at RT)spin max g, 10-15 min, 4ºCremove supernatant(alternative RNA precipitation - RNeasy from Qiagen) better than alcohol precipitation for smaller amounts of RNA (less risk of losing a miniscule nucleic acid pellet); also reduces risk of organic solvent contamination.
3. RNA wash:
A. 15-30 min depending on number of samples.
B. Wash pellet 70% EtOH (add & vortex briefly)70% ethanol prepared with RNase-free water some prefer to wash the pellet more than once with 70% ethanolspin max g, 2-10 min, 4°C air-dry pellet for 5-10 min Do not over dry the pellet or you won’t be able to re-dissolve it. Optional add RNase inhibitor.
C. Incubate at 55-60 C° for 10 min if hard to re-dissolve.
D. Transfer to Eppendorf tube spin 4°C, 5 min (to pellet undissolved material).
E. Re-dissolving of RNAdissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction)alternatively, 0.5% SDS.
F. Pipetting up and down, heat to 55-60°C for 10 min.
XIII.SDS/Polyacrylamide Gel Electrophoresis(CLICK)
Pour the Separating Gel
Set up your gel apparatus, prepare separating gel monomer. Add TEMED just prior to pouring gel. Allow to polymerize before adding stacking gel by overlaying gently with water or n-butanol. With higher concentrations of gels, one can immediately pour the stacking gel on the unpolymerized separating gel. Be careful not to mix the two layers. Separating Gels, in 0.375 M Tris, pH 8.8.
| 7% | 10% | 12% | 15% | |
| distilled H2O | 5.1 ml | 4.1 ml | 3.4 ml | 2.4 ml |
| 1.5 M Tris-HCl, pH 8.8 | 2.5 ml | 2.5 ml | 2.5 ml | 2.5 ml |
| 20% (w/v) SDS | 0.05 ml | 0.05 ml | 0.05 ml | 0.05 ml |
| Acrylamide/Bis-acrylamide(30%/0.8% w/v) | 2.3 ml | 3.3 ml | 4.0 ml | 5.0 ml |
| 10% (w/v) ammonium persulfate | 0.05 ml | 0.05 ml | 0.05 ml | 0.05 ml |
| TEMED | 0.005 ml | 0.005 ml | 0.005 ml | 0.005 ml |
| Total monomer | 10.005 ml | 10.005 ml | 10.005 ml | 10.005 ml |
Pour the Stacking Gel
After the separating gel has polymerized, decant the overlay, prepare the stacking monomer, add the TEMED, and pour. Insert the comb and allow to polymerize completely before running.
Stacking Gels, 4.0% gel, 0.125 M Tris, pH 6.8.
1. 3.075 ml distilled H2O
2. 1.25 ml 0.5 M Tris-HCl, pH 6.8
3. 0.025 ml 20% (w/v) SDS
4. 0.67 ml Acrylamide/Bis-acrylamide(30%/0.8% w/v)
5. 0.025 ml 10% (w/v) ammonium persulfate
6. 0.005 ml TEMED
7. 5.05 ml Total Stack monomer
For best results:
1. Make ammonium persulfate solution fresh daily.
2. Degas solutions before adding TEMED for 15 min at room temperature.
3. Running the gel
We usually run my gels at constant current, 25-50 mA, depending on gel size. Here"s the recipe for 5X SDS-PAGE running buffer. Dilute to 1X before use.
1. 1 liter 5X Running Buffer, pH 8.3
2. 15 g Tris Base
3. 72 g Glycine
4. 5 g SDS
5. distilled water to 1 liter
Store at room temperature until use.
Sample buffer
Dilute samples at least 1:4 with sample buffer, heat at 95 C for 4 minutes prior to loading. 8 ml Sample Buffer:
1. 4.0 ml Distilled water
2. 1.0 ml 0.5 M Tris-HCl
3. 0.8 ml Glycerol
4. 1.6 ml Acrylamide/Bis-acrylamide(30%/0.8% w/v)
5. 0.4 ml beta-mercaptoethanol
6. 0.2 ml bromophenol blue
XIV.Fluorescence detection(CLICK)
We use Fluoroskan Ascent FL according to: [PDF]Fluoroskan Ascent FL
XV.TAL effector construction(CLICK)
The construction of the TALEs were performed with the Golden Gate TALEN and TAL Effector Kit 2.0 by Addgene(R).
The protocol can be reached at : [PDF]TAL effector construction
XVI.Statistical analysis(CLICK)
Statistical evaluation of differences between means of experimental groups was done by ANOVAs and multiple Student tests. Both p values < 0.05 and p < 0.01 were considered to be significant. Data points represented the means of three independent experiments.
