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+ | <link rel="stylesheet" href="https://2017.igem.org/Team:CPU_CHINA/css/description?action=raw&ctype=text/css"> | ||
+ | </head> | ||
+ | <style> | ||
+ | #content{ | ||
+ | width: 100%; | ||
+ | } | ||
+ | h4{line-height:40px; | ||
− | <h1>Experiments</h1> | + | } |
− | < | + | h1,h2,h3,h4{ |
+ | font-family: "Century Gothic"; | ||
+ | } | ||
+ | </style> | ||
+ | <body> | ||
+ | <div class="body-container"> | ||
+ | <h1>Experiments</h1> | ||
+ | |||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>Western Blot</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Protocol</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> 1. Prepare Solutions | ||
+ | <br> 2. Gel Electrophoresis | ||
+ | <br> (1) Load protein and molecular weight marker | ||
+ | <br> (2) Add running buffer | ||
+ | <br> (3) Electrify: set the voltage at 80V before the sample reach the dividing line between stacking gel and resolving gel,switch to 130V until the blue stain reach the buttom line | ||
+ | <br>3. Blotting | ||
+ | |||
+ | <br><span style="font-weight:600">I. Transfer </span> | ||
+ | <br> (1)Carefully cut the gel | ||
+ | <br> (2)Assemble the transfer cassette | ||
+ | <br> (3)Install the cassette and electrify to transfer the protein to PVDF | ||
+ | |||
+ | <br><span style="font-weight:600">II. Chemiluminescene</span> | ||
+ | <br> (1) Blocking: 5% whole milk incubate for 1h | ||
+ | <br> (2) Incubate with diluted primary antibody | ||
+ | <br> (3) Wash membrane: TBST 15min *4 | ||
+ | <br> (4) Incubate with diluted secondary antibody | ||
+ | <br> (5) Wash membrane: TBST 15min *4 | ||
+ | <br> (6) Add Chemiluminescene substrate, incubate in dark for 5min | ||
+ | <br> (7) Exposure | ||
+ | |||
+ | </h4> | ||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>Immunoprecipitation(IP)</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Reagents : </h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> Lysis buffer | ||
+ | <br> Tris-HCl: 50 mM, pH 7.4 | ||
+ | <br> NP-40: 1% | ||
+ | <br> Na-deoxycholate: 0.5% | ||
+ | <br> NaCl: 150 mM | ||
+ | <br> COMPLETE protease inhibitors (Roche) | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Protocol : </h4> | ||
+ | <h4 > | ||
+ | <br> 1. Use 1 x 10 cm dish of near confluent cells for 1 ml of lysis buffer. | ||
+ | |||
+ | <br> 2. Wash cells in 2 ml ice cold PBS. | ||
+ | |||
+ | <br> 3. Lyse cells with 1 ml of 1x lysis buffer containing protease inhibitor cocktail (PIC). Wash cells off of plate | ||
+ | |||
+ | using a pipette or cell scraper. | ||
+ | |||
+ | <br> 4. Centrifuge at 4oC for 15 mins at 15000 rpm to pellet insoluble material. | ||
+ | |||
+ | <br> 5. Carefully remove supernatant to a fresh tube. | ||
+ | |||
+ | <br> 6. Add 1 μg of primary antibody and incubate with gentle mixing for 2-16 hours at 4oC. | ||
+ | |||
+ | <br> 7. Wash 20 ml of Protein G-sepharose in 1ml of lysis buffer | ||
+ | |||
+ | <br> 8. Resuspend in 100 μl and add to lysate containing antibody. | ||
+ | |||
+ | <br> 9. Incubate 1 hour with gentle mixing at 4oC. | ||
+ | |||
+ | <br> 10. Wash complexes 3 times in lysis buffer + PIC at 4oC. | ||
+ | |||
+ | <br> 11. Boil in 50 μl SDS sample buffer prior to loading on gel | ||
+ | |||
+ | <br> 12. Run on SDS-PAGE and immunoblot. | ||
+ | |||
+ | </h4> | ||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>Electrical transfection</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br><span class="dg">→ </span>Reagents : </h4> | ||
+ | <h4> | ||
+ | <br> DNA: 0.2 - 2.0µg/µl purified plasmid DNA | ||
+ | <br> Pipette glass: glass capillary tubing, boroscilicate, standard wall with filament (World Precision Instruments | ||
+ | Inc.) | ||
+ | <br> Electrode tip diameter will depend on the application. | ||
+ | |||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Protocol : </h4> | ||
+ | <h4> | ||
+ | <br> Micropipettes : A micropipette and Picospritzer are used to pressure inject DNA into the brain ventricle. The shape | ||
+ | and size of the pipette tip is not critical, but it must be sharp enough to easily pierce the tissue, and large | ||
+ | enough to quickly deliver the DNA. We use the Picospritzer II to deliver 75-125nl DNA solution directly into | ||
+ | the tadpole brain ventricle. The same pipette is used for multiple animals. | ||
+ | |||
+ | <br> DNA solution : We tested a range of plasmid concentrations (using Clontech pEGFP) and found that concentrations | ||
+ | ranging between 0.2 - 2.0µg/µl yield comparable numbers of fluorescent cells, with similar intensity of GFP fluorescence. | ||
+ | DNA can be diluted in dH2O, buffered saline, or 2mM CaCl2. The DNA solution was colored with 0.01% fast green | ||
+ | as a visual aid for filling the brain ventricle. For co-electroporation of two plasmids, we mix plasmids in a | ||
+ | ratio of 1:1. This typically gives a co-transfection rate of 70% ±10% (determined for the simultaneous electroporation | ||
+ | of of pEGFP and pDsRed). | ||
+ | |||
+ | <br> Setup:A dissecting microscope with good optics is sufficient. The micromanipulators are placed either side of | ||
+ | the stage. One manipulator holds the micropipette, connected to the Picospritzer. The other manipulator holds | ||
+ | the platinum electrodes connected to the capacitor and stimulator. | ||
+ | |||
+ | <br> Procedure : The anesthetized tadpole is placed on a moistened kimwipe on the center of the microscope stage. | ||
+ | The micropipette containing DNA is inserted into the ventricle of the tadpole brain, and the DNA is pressure | ||
+ | injected into the ventricle. For widespread electroporation, DNA is injected to fill the entire brain ventricle. | ||
+ | For targeted electroporation of a specific brain region, a concentrated bolus of DNA should be injected as close | ||
+ | as possible to the region of interest. The micropipette is removed, and the platinum electrodes are immediately | ||
+ | lowered to contact the tadpole's skin, spanning the brain region of interest (see Fig. 2). 2-7 voltage pulses | ||
+ | are delivered (depending on desired level of transfection). Effervescent bubbles are produced at the electrode | ||
+ | tips where they contact the skin. The level of effervescence is a good indicator of whether you have achieved | ||
+ | electroporation vs electrocution. There should be numerous small bubbles along the electrode tips. | ||
+ | <br>If the bubbles are large and bubbling over, the voltage is too large and the animal will die. Another visual | ||
+ | cue is the amount of shock the tadpole displays. The tadpole eyes usually flick in response to the electroporation. | ||
+ | If the whole body jolts, the voltage is too large. After electroporation, the tadpole is quickly returned to | ||
+ | rearing solution, where it usually recovers within 10 minutes. | ||
+ | |||
+ | <br> The DNA constructs can be targeted to just one side of the brain, or if desired, the whole brain can be transfected. | ||
+ | This is achieved by regulating the voltage polarity. If only one side of the brain is to be transfected, the | ||
+ | polarity setting on the stimulator is set so the negatively charged DNA moves towards the positive electrode. | ||
+ | If both sides of the brain are to be transfected, the polarity must be switched while the voltage pulses are | ||
+ | being delivered. | ||
+ | |||
+ | <br> Stimulation parameters : Depending on the number of transfection cells desired, 2 - 7 pulses of 30 - 50V with | ||
+ | an exponential decay of 70 ms are optimal. To transfect fewer cells, reduce the numbers of pulses. | ||
+ | |||
+ | <br> Detecting transfected cells : Transfected cells expressing GFP are detected by standard fluorescence microscopy. | ||
+ | |||
+ | <br> Trouble shooting : We occasionally see some bleeding in the brain ventricle 24 hrs after electroporation. This | ||
+ | usually clears up by 48 hrs. Propidium iodide staining indicated that electroporation does not cause an increase | ||
+ | in cell death. For good charge conduction: - Ensure that the specimen remains moist - The platinum electrodes | ||
+ | must be cleaned regularly. | ||
+ | |||
+ | </h4> | ||
+ | <h3 class="ar-title">Plasmid extraction</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br><span class="dg">→ </span>Reagents : </h4> | ||
+ | <h4> | ||
+ | <br> Solution I | ||
+ | <br> 50 mM glucose | ||
+ | <br> 25 mM Tris-Cl (pH 8.0) | ||
+ | <br> 10 mM EDTA (pH 8.0) | ||
+ | <br> Autoclave, and store at room temperature. | ||
+ | <br> | ||
+ | <br> Solution II | ||
+ | <br> 0.2N NaOH | ||
+ | <br> 1% SDS | ||
+ | <br> Store at room temperature in a plastic bottle. Don''t autoclave. | ||
+ | <br> | ||
+ | <br> Solution III | ||
+ | <br> 5M potassium acetate 60 ml | ||
+ | <br> glacial acetic acid 11.5 ml | ||
+ | <br> Distilled water 28.5 ml | ||
+ | <br> Store at room temperature. This solution can be autoclaved. However, we usually use this solution without autclaving. | ||
+ | <br> | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Protocol : </h4> | ||
+ | <h4> | ||
+ | <br>1.Shake E. coli harboring plasmid at 37 C overnight in 50 ml of TB containing appropriate antibiotics. (when using | ||
+ | ampicillin, addition of the antibiotics to 100-200 ug/ml rather than usual 50 ug/ml may improve the yield of | ||
+ | plasmids.) | ||
+ | |||
+ | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 min at 4 C. Discard the supernatant. | ||
+ | |||
+ | <br> 3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove | ||
+ | all of the supernatant fluid using pipetman. | ||
+ | |||
+ | <br> 4. Resuspend the pellet in 5 ml of Solution I. | ||
+ | |||
+ | <br> 5. Add 10 ml of Solution II. Mix well by inverting the bottle more than 10 times. The solution should be clear | ||
+ | after mixing Solution II. | ||
+ | |||
+ | <br> 6. Add 7.5 ml of Solution III. Mix well as above. | ||
+ | |||
+ | <br> 7. Spin the lysate at 10,000 rpm for 10 min at 4 C. | ||
+ | |||
+ | <br> 8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle | ||
+ | for 10 min at room temperature. | ||
+ | |||
+ | <br> 9. Spin at 10,000 rpm for 10 min at 4 C. Discard the supernatant. Remove all of the fluid using pipetman. | ||
+ | |||
+ | <br> 10. Dissolve the pellet in 600 ul of TE. Transfer the solution into a microfuge tube. | ||
+ | |||
+ | <br> 11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 min at 4 C. | ||
+ | |||
+ | <br> 12. Transfer the supernatant containing plasmid DNA to a new microfuge tube. Add 600 ul of isopropanol. Mix | ||
+ | well, and then spin the solution at 14,000 rpm for 5 min at 4 C. | ||
+ | |||
+ | <br> 13. Discard the supernatant. Rince the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard | ||
+ | the fluid. | ||
+ | |||
+ | <br> 14. Add to the pellet 400 ul of TE containing DNase-free RNase A (20 ug/ml). Incubate the tube for 30 min at | ||
+ | 37 C. | ||
+ | |||
+ | <br> 15. After 30 min, carefully check the content of the tube. If nucleic acid pellet is visible at the bottom of | ||
+ | the tube, vortex well to dissolve the pellet. Incubate the tube at 37 C for further 30 min. | ||
+ | |||
+ | <br> 16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent | ||
+ | to PEG8000, and works well.) | ||
+ | |||
+ | <br> 17. Spin at 14,000 rpm for 5 min. Discard the supernatant. Rinse the pellet with 300 ul of cold 70% ethanol. | ||
+ | Discar the fluid. Dissolve the pellet in 400 ul of TE. (Optional: Repeat PEG precipitation once more. This is | ||
+ | recommended for preparing dephosphorylated linear vector since trace amount of short RNA in the vector preparation | ||
+ | may interfere with the dephosphorylation reaction.) | ||
+ | |||
+ | <br> 18. Extract the plasmid solution with chloroform to remove PEG. Take aquaous phase. Extract the aquaous phase | ||
+ | with phenol. Take aquaous phase. (Optional: Repeat phenol extraction until no interphase is visible. This is | ||
+ | recommended for preparing the template for in vitro transcription.) | ||
+ | |||
+ | <br> Extract the aquaous phase with chloroform or ethylether to remove trace amount of phenol dissolved in the solution. | ||
+ | Take aquaous phase (or remove organic phase). | ||
+ | |||
+ | <br> 19. Add 0.1 volume of 3 M sodium acetate and 3 volume of ethanol into the plasmid solution. Spin at 14,000 rpm | ||
+ | for 5 min at 4 C. Discard the supernatant. Rinse the pellet with 200 - 500 ul of 70% ethanol. | ||
+ | |||
+ | <br> 20. Store the open tube on the bench until the visible traces of ethanol have evaporated. | ||
+ | |||
+ | <br> 21. Dissolve the DNA pellet in 200-500 ul of TE. Typically 300-800 ug of plasmid should be obtained if the plasmid | ||
+ | have pUC-based replication origin. | ||
+ | |||
+ | |||
+ | </h4> | ||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>Lentivirus packaging</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Production of Lentiviral Vectors: Packaging of the Vectors :</h4> | ||
+ | <h4> | ||
+ | <br> 1.Maintain 293T cells in complete culture medium in a 37°C incubator with 5% CO2. Twenty-four hours before transfection, | ||
+ | plate exponentially growing 293T cells in 100-mm tissue culture dishes at 4 x 106 cells/plate. | ||
+ | Cell density should be approximately 80% confluent for transfection. | ||
+ | |||
+ | <br>2.Prepare 1 ml of calcium phosphate-DNA suspension for each 100-mm plate of cells as follows: | ||
+ | |||
+ | <br>3.Set up two sterile tubes for transfection of one plate. Label the tubes 1 and 2. | ||
+ | |||
+ | <br>4.Add 0.5 ml of 2x HBS to Tube 1. | ||
+ | |||
+ | <br>5.Add TE 79/10 to Tube 2. The volume of TE 79/10 = 440 µl minus the volume of the DNA solution. | ||
+ | |||
+ | <br>6.Add 15 µg of the transfer vector containing the transgene, 15 µg of pCgp, 5 µg of pCMV-rev, | ||
+ | and 5 µg of pCMV-G to Tube 2 and mix. | ||
+ | |||
+ | <br>7.Add 60 µl of 2 M CaCl2 solution to Tube 2 and mix gently. | ||
+ | |||
+ | <br>8.Transfer the contents from Tube 2 to Tube 1 dropwise with gentle mixing. | ||
+ | |||
+ | <br>9.Allow the suspension to sit for 30 minutes at room temperature. | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Mix the precipitate well by pipetting or vortexing </h4> | ||
+ | <h4> | ||
+ | <br> 1.Add 1 ml of the suspension to a 100-mm plate containing cells. Add the suspension slowly, | ||
+ | dropwise while gently swirling the medium in the plate. Return the plates to the 37°C incubator and | ||
+ | leave the precipitate for 4 hours. | ||
+ | |||
+ | <br>2.Replace the old medium with 6 ml of fresh culture medium. Add 60 µl of 0.6 M sodium butyrate. Return to the incubator. | ||
+ | |||
+ | <br>3.After 48 hours of culture, collect the supernatant and freeze it at -80°C or proceed to the concentration step. | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Concentration of the Vectors </h4> | ||
+ | <h4> | ||
+ | <br> 1.Centrifuge the supernatant (freshly collected or thawed from the freezer) at 900g for 10 minutes to | ||
+ | remove any cell debris in the supernatant. | ||
+ | |||
+ | <br>2.Filter the supernatant through a 0.2-µm syringe filter. | ||
+ | |||
+ | <br> 3.Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation | ||
+ | for 1.5 hours at 4°C in a Beckman SW 28 swinging bucket rotor at 24,500 rpm. | ||
+ | |||
+ | <br> 4.Remove the supernatant and resuspend the pellet in an appropriate amount of culture medium, e.g., | ||
+ | 300 µl for 30 ml of original supernatant if a 100-fold concentration is desired. | ||
+ | |||
+ | <br> 5.Divide the concentrated vector into 10-50-µl aliquots and store at -80°C until use. | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Titration of the Vectors </h4> | ||
+ | <h4> | ||
+ | <br> 1.Seed 5 x 104 HT1080 cells/well in a 12-well plate in complete medium and culture overnight in a 37°C incubator with 5% CO2. | ||
+ | |||
+ | <br> 2.Add serial diluted vector stock and 4 µl/ml polybrene to the cultured cells. Continue culture for 48 hours. | ||
+ | |||
+ | <br> 3.Trypsinize the cells. Following centrifugation, remove the supernatant and resuspend the pellet in 300 µl of 3.7% formaldehyde in PBS. | ||
+ | |||
+ | <br> 4.Determine the percentage of EGFP-positive cells by FACS analysis. | ||
+ | |||
+ | <br> 5.The titer will be represented as transduction units (TUs) per milliliter concentrated vector (TU/ml). | ||
+ | </h4> | ||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>Lentivirus transfection</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br><span class="dg">→ </span>Transduction of Lentiviral Vectors to Target Cells</h4> | ||
+ | <h4> | ||
+ | <br> 1.Seed exponentially growing cells at 2 x 105 cells/well in 1 ml of culture medium into a 24-well plate. | ||
+ | |||
+ | <br>2.Add various amounts of concentrated vector stock depending on cell type. | ||
+ | |||
+ | <br>3.Add 4 µg/ml polybrene. Return the cells to a 37°C incubator. | ||
+ | |||
+ | <br> 4.After overnight incubation, centrifuge the cells, discard the supernatant, and resuspend | ||
+ | the cells with fresh culture medium. Return the cells to culture. | ||
+ | |||
+ | <br> 5.Determine transduction efficiency by FACS analysis 48 hours after transduction (see Troubleshooting). | ||
+ | |||
+ | <br> 6.Purify CD34+ hematopoietic stem cells from umbilical cord blood or bone marrow using anti-CD34 | ||
+ | antibody-coupled magnetic beads following the manufacturer's protocol. | ||
+ | |||
+ | <br> 7.Forty-eight hours before transduction, culture the CD34+ cells in CD34+ transduction medium. | ||
+ | <br> 8.Coat a 48-well nontissue-culture-treated plate with 0.2 ml of 25 µg/ml RetroNectin (~5 µg/cm2) for 2 hours at room temperature | ||
+ | |||
+ | <br> 9.Remove RetroNectin and then add 0.2 ml of 2% BSA in PBS for blocking. Store the plate for 30 minutes at room temperature. | ||
+ | |||
+ | <br> 10.After washing the wells with PBS, adjust the lentiviral vector stock to the appropriate moi (range 5-40) | ||
+ | with plain IMDM medium to 200-µl volume and load it into the well of the coated plate. | ||
+ | |||
+ | <br> 11.After incubation for 2 hours at 37°C, remove the vector supernatant and then wash the well with PBS. | ||
+ | |||
+ | <br> 12.Add the prestimulated CD34+ cells to the well at 1 x 105 cells/well in 0.2 ml of growth medium and | ||
+ | return the cells to the 37°C incubator. | ||
+ | |||
+ | <br> 13.After overnight culture, centrifuge the cells, resuspend the cell pellet in 1 ml of culture medium, | ||
+ | and transfer the cells to a 24-well plate. Return the cells to the incubator. | ||
+ | |||
+ | <br> 14.Determine transduction efficiency by FACS analysis 6 days after transduction. | ||
+ | </h4> | ||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>qPCR</h3> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Reagents:</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> dNTP mixture (TaKaRa,4030); | ||
+ | |||
+ | <br> Taq (TaKaRa,DR100A); | ||
+ | |||
+ | <br> GAPDH probe; | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600"> | ||
+ | <br> <span class="dg">→ </span>Protocol:</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> 1. Components: total volume 20μL | ||
+ | |||
+ | <br> 10×buffer 2μL dNTPs mixture (10mmol) 0.4μL MgCl 2 1.2μL Taq 0.2μL Sense primer (10mM) 0.5μL Antisense primer (10mM) 0.5μL ddH O 13.1μL DNA 1μL | ||
+ | |||
+ | <br> 2. Cycling Conditions: | ||
+ | |||
+ | <br> Step 1: 95℃, 5min | ||
+ | |||
+ | <br> Step 2: 95℃, 30s | ||
+ | |||
+ | <br> Step 3: 95℃, 30s | ||
+ | |||
+ | <br> Step 4: 95℃, 30s (fluorescence detection) | ||
+ | |||
+ | <br> Step2-Step4, 40 cycles (variable, can be up to 45 cycles) | ||
+ | |||
+ | <br> 3. Data Analysis: The Comparative Ct Method (ΔΔCT Method) | ||
+ | |||
+ | <br> CT---cycles when the reaction reach the threshold, the relative expression level of each miRNA | ||
+ | compares to endogenous Control can be described as 2-ΔCT, (ΔCT= CT sample- CT endogenous control). | ||
+ | GAPDH, a housekeeping gene, is usually used as endogenous Control for mRNA. | ||
+ | </h4> | ||
+ | <h3 class="ar-title" style="margin-top:20px;"> | ||
+ | <span class="dg"> </span>RNA extraction using TRIzol/TRI</h3> | ||
+ | <h4 style="font-weight:600;margin-top:20px;"> | ||
+ | <br> <span class="dg">→ </span>Reagents:</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> TRIzol or TRI reagent | ||
+ | <br>0.8 M sodium citrate / 1.2 M NaCl | ||
+ | <br>isopropanol (2-propanol) | ||
+ | |||
+ | <br>chloroform | ||
+ | |||
+ | <br>75% EtOH in DEPC H2O | ||
+ | |||
+ | <br>RNase free water (filtered or DEPC) | ||
+ | </h4> | ||
+ | <h4 style="font-weight:600;margin-top:20px;"> | ||
+ | <br> <span class="dg">→ </span>Protocol:</h4> | ||
+ | <h4 ><br>1.cell lysis</h4> | ||
+ | <h4 style="padding-left:30px;margin-top:0px;"> | ||
+ | <br>Cell lysis only takes a few minutes per well, but tissue homogenisation can take 10-20 minutes per sample depending on how tough the tissue is. | ||
+ | |||
+ | <br>(1) (PBS wash) | ||
+ | |||
+ | <br> (2) add trizol (cell lysis) | ||
+ | |||
+ | <br> 1ml / 3.5 cm diameter well (6-well) | ||
+ | |||
+ | <br> 5ml / 75 ml bottle | ||
+ | |||
+ | <br> (3) homogenise by pipetting several times (mechanic lysis) | ||
+ | |||
+ | <br> alternative for tubes: vortex 1 min | ||
+ | |||
+ | <br> alternative for tissue: grind 1 g tissue in liquid nitrogen in a motar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 min at room temp or 60° C (scaled up as needed) | ||
+ | |||
+ | <br> (4) (5min at RT for complete dissociation of nucleoprotein complexes) | ||
+ | |||
+ | <br>RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample | ||
+ | in trizol for a short time or freezing it for a longer one. | ||
+ | </h4> | ||
+ | <h4> <br>2.phase separation</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br>15-45 min depending on number of samples and whether an additional chloroform wash is necessary | ||
− | < | + | <br> (1) add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml) |
− | + | ||
− | </ | + | <br> (2) shake for 15 sec (Eccles protocol: do not vortex) |
+ | |||
+ | <br> (3) incubate 2-5 min at RT | ||
+ | |||
+ | <br> (4) spin max. 12000g, 5-15 min, 2-8°C | ||
+ | |||
+ | <br> if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted | ||
+ | |||
+ | <br> If supernatant appears turbid an additional chloroform cleaning step can be inserted here. | ||
+ | |||
+ | <br> (5) transfer aqueous upper phase into new tube | ||
+ | |||
+ | <br>Take care not to aspirate the DNA-containing white interface. This quickly happens and will lead to DNA contamination in your RNA prep. | ||
+ | |||
+ | <br> TRIZOL phases after chloroform addition | ||
+ | |||
+ | <br> TOP - colourless aqueous phase (RNA) - 60% TRIZOL volume | ||
+ | |||
+ | <br> MIDDLE - interphase (DNA) | ||
+ | |||
+ | <br> BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids) | ||
+ | </h4> | ||
+ | <h4 > <br>3.RNA precipitation and wash</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br>20-40 min depending on number of samples | ||
+ | |||
+ | <br>(1) add isopropanol (70% of aqueous phase or 1/2 trizol volume) | ||
+ | |||
+ | <br>(2) 0.8 M sodium citrate or 1.2 M NaCl can be added | ||
+ | |||
+ | <br>(3) (incubate 10min at RT) | ||
+ | |||
+ | <br>(4) spin max g, 10-15 min, 4ºC | ||
+ | |||
+ | <br>(5) remove supernatant | ||
+ | |||
+ | <br>(6) (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 | ||
+ | |||
+ | <br>similar kits to RNeasy: MinElute kit, or Affymetrix sample clean-up | ||
+ | </h4> | ||
+ | <h4 > <br>4.RNA wash</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> 15-30 min( depending on number of samples) | ||
+ | |||
+ | <br>(1) wash pellet 70% EtOH (add & vortex briefly) | ||
+ | |||
+ | <br> 70% ethanol prepared with RNase-free water | ||
+ | |||
+ | <br>some prefer to wash the pellot more than once with 70% ethanol | ||
+ | |||
+ | <br>(2) spin max g, 2-10 min, 4ºC | ||
+ | |||
+ | <br> (3) air-dry pellet for 5-10 min Do not overdry the pellet or you won''t be able to redissolve it. | ||
+ | |||
+ | <br> optional add RNase inhibitor | ||
+ | |||
+ | <br> (4) incubate at 55-60 C° for 10 min if hard to redissolve | ||
+ | |||
+ | <br>(5) transfer to eppendorf tube | ||
+ | |||
+ | <br> (6) spin 4° C, 5 min (to pellet undissolved material) | ||
+ | </h4> | ||
+ | <h4 ><br>5.redissolving of RNA</h4> | ||
+ | <h4 style="padding-left:30px;"> | ||
+ | <br> (1) dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction) | ||
+ | |||
+ | <br>(2) alternatively, 0.5% SDS, pipetting up and down, heat to 55-60°C for 10 min | ||
+ | </h4> | ||
+ | </div> | ||
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+ | </body> | ||
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Revision as of 16:43, 31 October 2017