NORTHERN BLOT |
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Last Update: December 2006 |
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This protocol is based on the protocols developed in Pamela Green's laboratory |
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| PREPARE SOLUTIONS | |
| 1. 20X MOPS buffer (2 L): | Mix 167.45 g of MOPS, 16.4 g of NaOAc, 80 mL of 0.5M EDTA, pH to 7.0 with NaOH, add dH2O to 2 L (store in the dark) |
| 2. 1X MOPS running buffer (1 L): | Mix 100 mL of 20X MOPS with 900 mL of dH2O. Add 10 mL of 10 mg/mL EtBr before use. |
| 3. Forlmaldehyde gel dye: | Mix 3.6 mL of Deionized formaldehyde, 0.4 mL of 20X MOPS, 1.3 mL of Formaldehyde solution, 1.3 mL of DEPC-dH2O, 0.5 mL of 80% Glycerol, and Bromophenol Blue to taste (sprinkle) |
| 4. 10 mg/mL Salmon Sperm DNA: | Mix 100 mg of Salmon Sperm DNA with 10 mL of dH2O. Store at -20oC (BOIL before use) |
| 5. Pre- and Hybridization solution (random primed probe) 25 mL: | Mix 10.75 mL of DEPC-dH2O, 6.25 mL of 20X SSC, 5 mL (10X) of 50X Denhardts, 2.5 mL (0.1 M) of 1M K phosphate pH 6.8, 0.25 mL (100 ug/mL) (freshly boiled) of 10 mg/mL Salmon Sperm DNA, and 0.25 mL (0.2%) of 20% SDS. |
| 6. 20X SSPE (1 l): | Mix 175.3 g of NaCl, 27.6 g of NaH2PO4-H2O, 7.4 g of EDTA, 800 mL of dH2O, pH to 7.4 w/ NaOH, dH2O to 1 L (Autoclave) |
| 7. 1 M KH2PO4 (monobasic): | Mix 13.6 g of KH2PO4 with 100 mL of dH2O (Autoclave) |
| 8. 1 M K2HPO4 (dibasic): | Mix 17.42 g of K2HPO4 with 100 mL of dH2O (Autoclave) |
| 9. DEPC-dH2O (1 l): | Mix 1 L of dH2O with 1 mL of DEPC. Stirr at 37oC ON (Autoclave) |
| 10. 20X SSC (2 L): | Mix 348 g of NaCl with 176.4 g of Na-Citrate-2H2O. pH to 7.0
and dH2O to 2 L |
| 11. 10 mg/mL EtBr (10 mL): | Mix 100 mg of EtBr with 10 mL of dH2O |
| 12. 50X Denhardts (500 mL): | Mix 5 g of PVP-360, 5 g of Ficoll-400, 5 g of BSA (Fraction V), dH2O to 500 mL. Slow mix at 30oC. Filter and sotre at -20oC |
| 13. Pre- and Hybridization soln (riboprobe) 10 mL: | Mix 5 mL (50%) of Formamide, 2.5 mL (5X) of 20X SSPE, 2 mL (10X) of 50X Denhardts, 0.1 mL (freshly boiled) of 10 mg/mL Salmon Sperm DNA, 0.1 mL (0.2%) of 20% SDS, 0.3 mL of DEPC-dH2O |
| 14. 80% Glycerol (100 mL): | Mix 80 mL of Glycerol with 20 mL of dH2O (Autoclave) |
| 15. 20% SDS (1 L): | Mix 200 g of SDS, 800 mL of dH2O, heat to 68oC, pH to 7.2 w/ HCl and add
dH2O to 1 L |
| 16. 1M K phosphate pH 6.8 | Mix 50.3 mL of 1M KH2PO4 with 49.7 mL of 1M K2HPO4 |
| 17. 0.5 M EDTA (pH 8.0): | Mix 186.1 g of disodium ethylenediaminetetraacetate-2H2O with 800 mL dH2O. pH to 8.0 with NaOH (~20 g) (Autoclave) |
| PROCEDURE | |
| 1. Weigh out 1.1 g agarose and place in an 500 mL Erlenmeyer flask (volume is higher to acount for evaporation) | |
| 2. Add 5 mL 20X MOPS buffer, 92.5 mL DEPC-dH2O and microwave until melted. Stir to mix | |
| 3. Place in 65oC waterbath for 5-10 minutes | |
4. Set up RNA gel tray in fume hood, add 1 mL of 10 mg/mL EtBr and 5.4 mL of formaldehyde solution to the gel and swirl to mix throughly |
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| 5. Pour gel and let polymerize (~20 mins) and place of gel tank with 1X MOPS running buffer. Remove comb. | |
| 6. Once RNA is dry (SpeedVac) resuspend in 20 mL Forlmaldehyde gel dye and vortex for 5 mins, incubate for 5 mins at 65oC, quick cool ice and load samples. Run gel at 90-110 V for 3-4 hrs | |
| 7. Gel setup: | |
| 7.1. pre-wet Nylon membrane in DEPC- dH2O and then in 5X SSC | |
7.2. Place a large 3MM Whatman paper on top of wick (soaked with 10X SSC) |
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| 7.3. Place place 3MM Whatman papers (2-3) cut to the size of the gel on top of wick and then place gel, previously washed for 20 mins in 10X SSC (place face down on Northern setup) | |
| 7.4. Place pre-wet Nylon membrane (Hybond-N) on top of gel and then place 2 5XSSC pre-wet Whatman papers on top of membrane | |
| 7.5. Finally put paper towels and a piece of glass (to distribute the weight evenly) plus a ~1 kg weigth on top | |
8. Let transfer proceed ON |
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9. Wash membrane in 2X SSC for 2-5 mins |
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10. UV-crosslink RNA to membrane using a UV-Stratlinker (Autocrosslink option). Keep membrane moist at all times |
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11. Gel can be stored dried or used immediately |
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12. If dry, pre-wet gel with 5X SSC. Add prehybridization solution (~15 mL) and incubate for >4 hrs at 50-65oC |
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13. During Prehybridization, label and purify probe |
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14. Add 500,000-1 million CPM of probe / mL of hybridization buffer (~15 mL) |
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15. Hybridize ON at 51-65oC |
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16. Wash tringency: |
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16.1. High: 3X 15 mins in 50 mL 0.2X SSC, 0.5% SDS at 65oC |
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16.2. Moderate: 3X 15 mins in 50 mL 3X SSC, 0.1% SDS at RT, 3X 20 mins in 3X SSC, 0.1% SDS at 50-55oC |
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NOTE: Monitor radioactivity of blots and washes |
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17. Remove membrane and blot slightly on 3MM Whatmam paper, place on a seal-a-meal bag and expose |
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NOTE: Expose at -70oC if using film, at room temperature if using a phosphoimager cassette |
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18. Strip probe from membrane: boil 0.1X SSC, 0.1% SDS, pour on membrane and let cool to RT while shacking. If necessary, repeat twice. Air dry membrane and store at RT |
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