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− | < | + | <h5>Experiments</h5> |
<h3 class="ar-title" style="margin-top:20px;"> | <h3 class="ar-title" style="margin-top:20px;"> | ||
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<br> (1) Blocking: 5% whole milk incubate for 1h | <br> (1) Blocking: 5% whole milk incubate for 1h | ||
<br> (2) Incubate with diluted primary antibody | <br> (2) Incubate with diluted primary antibody | ||
− | <br> (3) Wash membrane: TBST | + | <br> (3) Wash membrane: TBST 15 mins *4 |
<br> (4) Incubate with diluted secondary antibody | <br> (4) Incubate with diluted secondary antibody | ||
− | <br> (5) Wash membrane: TBST | + | <br> (5) Wash membrane: TBST 15 mins *4 |
− | <br> (6) Add Chemiluminescene substrate, incubate in dark for | + | <br> (6) Add Chemiluminescene substrate, incubate in dark for 5 mins |
<br> (7) Exposure | <br> (7) Exposure | ||
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using a pipette or cell scraper. | using a pipette or cell scraper. | ||
− | <br> 4. Centrifuge at | + | <br> 4. Centrifuge at 4℃ for 15 mins at 15000 rpm to pellet insoluble material. |
<br> 5. Carefully remove supernatant to a fresh tube. | <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 | + | <br> 6. Add 1 μg of primary antibody and incubate with gentle mixing for 2-16 hours at 4℃. |
<br> 7. Wash 20 ml of Protein G-sepharose in 1ml of lysis buffer | <br> 7. Wash 20 ml of Protein G-sepharose in 1ml of lysis buffer | ||
− | <br> 8. Resuspend | + | <br> 8. Resuspend to 100 μl and add to lysate containing antibody. |
− | <br> 9. Incubate 1 hour with gentle mixing at | + | <br> 9. Incubate 1 hour with gentle mixing at 4℃. |
− | <br> 10. Wash complexes 3 times in lysis buffer + PIC at | + | <br> 10. Wash complexes 3 times in lysis buffer + PIC at 4℃. |
<br> 11. Boil in 50 μl SDS sample buffer prior to loading on gel | <br> 11. Boil in 50 μl SDS sample buffer prior to loading on gel | ||
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<br> <span class="dg">→ </span>Protocol : </h4> | <br> <span class="dg">→ </span>Protocol : </h4> | ||
<h4> | <h4> | ||
− | <br> Micropipettes : A micropipette and Picospritzer are used to pressure | + | <br> Micropipettes : A micropipette and Picospritzer are used to pressure injected 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 | 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 | 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. | the tadpole brain ventricle. The same pipette is used for multiple animals. | ||
− | <br> DNA solution : We | + | <br> DNA solution : We test a wide range of plasmid concentrations (using Clontech pEGFP) and find that concentrations |
ranging between 0.2 - 2.0µg/µl yield comparable numbers of fluorescent cells, with similar intensity of GFP fluorescence. | 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 | DNA can be diluted in dH2O, buffered saline, or 2mM CaCl2. The DNA solution was colored with 0.01% fast green | ||
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of of pEGFP and pDsRed). | of of pEGFP and pDsRed). | ||
− | <br> | + | <br> Set-up:A dissecting microscope with good optics is imperative. The micro-manipulators are placed either side of |
− | the stage. One manipulator holds the micropipette, | + | the stage. One manipulator connected to the Picospritzer holds the micropipette, and the other one holds |
the platinum electrodes connected to the capacitor and stimulator. | 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. | <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 | + | The micropipette containing DNA is inserted into the ventricle of the tadpole brain, and the DNA is injected into the ventricle under pressure. 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 | 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 | as possible to the region of interest. The micropipette is removed, and the platinum electrodes are immediately | ||
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tips where they contact the skin. The level of effervescence is a good indicator of whether you have achieved | 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. | electroporation vs electrocution. There should be numerous small bubbles along the electrode tips. | ||
− | <br>If the bubbles are large and bubbling over, the voltage | + | <br>If the bubbles are large and bubbling over, the voltage will be too high 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. | 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 | + | If the whole body jolts, the voltage must be too large. After electroporation, the tadpole is quickly returned to |
rearing solution, where it usually recovers within 10 minutes. | 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 | + | <br> The DNA constructs can be targeted to just one side of the brain, or if possible, 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 | 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. | polarity setting on the stimulator is set so the negatively charged DNA moves towards the positive electrode. | ||
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<br> Stimulation parameters : Depending on the number of transfection cells desired, 2 - 7 pulses of 30 - 50V with | <br> Stimulation parameters : Depending on the number of transfection cells desired, 2 - 7 pulses of 30 - 50V with | ||
− | an exponential decay of 70 ms | + | an exponential decay of 70 ms is optimal. To transfect fewer cells, we should reduce the numbers of pulses. |
<br> Detecting transfected cells : Transfected cells expressing GFP are detected by standard fluorescence microscopy. | <br> Detecting transfected cells : Transfected cells expressing GFP are detected by standard fluorescence microscopy. | ||
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<br> 0.2N NaOH | <br> 0.2N NaOH | ||
<br> 1% SDS | <br> 1% SDS | ||
− | <br> Store at room temperature in a plastic bottle. Don | + | <br> Store at room temperature in a plastic bottle. Don't autoclave. |
<br> | <br> | ||
<br> Solution III | <br> Solution III | ||
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plasmids.) | plasmids.) | ||
− | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 | + | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 mins at 4℃. Discard the supernatant. |
<br> 3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove | <br> 3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove | ||
− | all of the supernatant | + | all of the liquid supernatant using pipetman. |
<br> 4. Resuspend the pellet in 5 ml of Solution I. | <br> 4. Resuspend the pellet in 5 ml of Solution I. | ||
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<br> 6. Add 7.5 ml of Solution III. Mix well as above. | <br> 6. Add 7.5 ml of Solution III. Mix well as above. | ||
− | <br> 7. Spin the lysate at 10,000 rpm for 10 | + | <br> 7. Spin the lysate at 10,000 rpm for 10 mins at 4℃. |
<br> 8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle | <br> 8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle | ||
− | for 10 | + | for 10 mins at room temperature. |
− | <br> 9. Spin at 10,000 rpm for 10 | + | <br> 9. Spin at 10,000 rpm for 10 mins at 4℃. 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> 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 | + | <br> 11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 mins at 4℃. |
<br> 12. Transfer the supernatant containing plasmid DNA to a new microfuge tube. Add 600 ul of isopropanol. Mix | <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 | + | well, and then spin the solution at 14,000 rpm for 5 mins at 4℃. |
− | <br> 13. Discard the supernatant. | + | <br> 13. Discard the supernatant. Rinse the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard |
the fluid. | 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 | + | <br> 14. Add to the pellet 400 ul of TE containing DNase-free RNase A (20 ug/ml). Incubate the tube for 30 mins at |
37 C. | 37 C. | ||
− | <br> 15. After 30 | + | <br> 15. After 30 mins, 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 | + | the tube, vortex well to dissolve the pellet. Incubate the tube at 37°C for further 30 mins. |
<br> 16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent | <br> 16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent | ||
to PEG8000, and works well.) | to PEG8000, and works well.) | ||
− | <br> 17. Spin at 14,000 rpm for 5 | + | <br> 17. Spin at 14,000 rpm for 5 mins. Discard the supernatant. Rinse the pellet with 300 ul of cold 70% ethanol. |
− | + | Discard 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 | recommended for preparing dephosphorylated linear vector since trace amount of short RNA in the vector preparation | ||
may interfere with the dephosphorylation reaction.) | may interfere with the dephosphorylation reaction.) | ||
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<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 | <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 | + | for 5 mins at 4℃. 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> 20. Store the open tube on the bench until the visible traces of ethanol have evaporated. | ||
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<h4> | <h4> | ||
<br> 1.Maintain 293T cells in complete culture medium in a 37°C incubator with 5% CO2. Twenty-four hours before transfection, | <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. | + | 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>2.Prepare 1 ml of calcium phosphate-DNA suspension for each 100-mm plate of cells as follows: | ||
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<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>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 - | + | <br>3.After 48 hours of culture, collect the supernatant and freeze it at -80℃ or proceed to the concentration step. |
</h4> | </h4> | ||
<h4 style="font-weight:600"> | <h4 style="font-weight:600"> | ||
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<br> 3.Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation | <br> 3.Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation | ||
− | for 1.5 hours at | + | for 1.5 hours at 4℃ 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., | <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. | 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 - | + | <br> 5.Divide the concentrated vector into 10-50-µl aliquots and store at -80℃ until use. |
</h4> | </h4> | ||
<h4 style="font-weight:600"> | <h4 style="font-weight:600"> | ||
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<br> 2. Cycling Conditions: | <br> 2. Cycling Conditions: | ||
− | <br> Step 1: 95℃, | + | <br> Step 1: 95℃, 5mins |
<br> Step 2: 95℃, 30s | <br> Step 2: 95℃, 30s | ||
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<br> alternative for tubes: vortex 1 min | <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 | + | <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 mins at room temp or 60° C (scaled up as needed) |
− | <br> (4) ( | + | <br> (4) (5 mins 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 | <br>RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample | ||
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<h4> <br>2.phase separation</h4> | <h4> <br>2.phase separation</h4> | ||
<h4 style="padding-left:30px;"> | <h4 style="padding-left:30px;"> | ||
− | <br>15-45 | + | <br>15-45 mins 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> (1) add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml) | ||
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<br> (2) shake for 15 sec (Eccles protocol: do not vortex) | <br> (2) shake for 15 sec (Eccles protocol: do not vortex) | ||
− | <br> (3) incubate 2-5 | + | <br> (3) incubate 2-5 mins at RT |
− | <br> (4) spin max. 12000g, 5-15 | + | <br> (4) spin max. 12000g, 5-15 mins, 2-8°C |
<br> if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted | <br> if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted | ||
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<h4 > <br>3.RNA precipitation and wash</h4> | <h4 > <br>3.RNA precipitation and wash</h4> | ||
<h4 style="padding-left:30px;"> | <h4 style="padding-left:30px;"> | ||
− | <br>20-40 | + | <br>20-40 mins depending on number of samples |
<br>(1) add isopropanol (70% of aqueous phase or 1/2 trizol volume) | <br>(1) add isopropanol (70% of aqueous phase or 1/2 trizol volume) | ||
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<br>(2) 0.8 M sodium citrate or 1.2 M NaCl can be added | <br>(2) 0.8 M sodium citrate or 1.2 M NaCl can be added | ||
− | <br>(3) (incubate | + | <br>(3) (incubate 10 mins at RT) |
− | <br>(4) spin max g, 10-15 | + | <br>(4) spin max g, 10-15 mins, 4℃ |
<br>(5) remove supernatant | <br>(5) remove supernatant | ||
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<h4 > <br>4.RNA wash</h4> | <h4 > <br>4.RNA wash</h4> | ||
<h4 style="padding-left:30px;"> | <h4 style="padding-left:30px;"> | ||
− | <br> 15-30 | + | <br> 15-30 mins( depending on number of samples) |
<br>(1) wash pellet 70% EtOH (add & vortex briefly) | <br>(1) wash pellet 70% EtOH (add & vortex briefly) | ||
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<br>some prefer to wash the pellot more than once with 70% ethanol | <br>some prefer to wash the pellot more than once with 70% ethanol | ||
− | <br>(2) spin max g, 2-10 | + | <br>(2) spin max g, 2-10 mins, 4℃ |
− | <br> (3) air-dry pellet for 5-10 | + | <br> (3) air-dry pellet for 5-10 mins Do not overdry the pellet or you won''t be able to redissolve it. |
<br> optional add RNase inhibitor | <br> optional add RNase inhibitor | ||
− | <br> (4) incubate at 55-60 C° for 10 | + | <br> (4) incubate at 55-60 C° for 10 mins if hard to redissolve |
<br>(5) transfer to eppendorf tube | <br>(5) transfer to eppendorf tube | ||
− | <br> (6) spin | + | <br> (6) spin 4℃, 5 mins (to pellet undissolved material) |
</h4> | </h4> | ||
<h4 ><br>5.redissolving of RNA</h4> | <h4 ><br>5.redissolving of RNA</h4> | ||
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<br> (1) dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction) | <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 | + | <br>(2) alternatively, 0.5% SDS, pipetting up and down, heat to 55-60°C for 10 mins |
</h4> | </h4> | ||
</div> | </div> |
Latest revision as of 15:19, 1 November 2017