contributed by James McCaughern-Carucci, Yale University
Resuspending RNA in Formamide (as reported by Chomczynski et al. Nucleic Acids Research) has several benefits over storage in water or ethanol. First, formamide will protect RNA from RNases. The RNA is also extremely stable, allowing it to be stored overnight at 4C, or indefinitely at -20C. Samples can even be left at room temperature overnight without fear of degradation. Samples can be made to be highly concentrated, up to 4 mg/ml. Finally, with concentrated samples, no drying of your RNA is necessary for running Northerns, RT-PCR, or RNase Protection, saving time and potential degradation.
Precipitate your RNA as usual with either 100% ETOH (CsCl method) or Isopropanol (other methods). Spin to pellet.
Wash RNA pellet with 75% ETOH to remove excess salt...especially if using CsCl !!!
Aspirate ETOH, and make sure pellet is free of excess of ETOH, but DO NOT OVERDRY!!
Resuspend pellet in 100% formamide, which has been stored at 4C. Resuspend your pellet in anywhere from 10ul to 1000ul, depending upon pellet size.
Most or all of the pellet should dissolve instantly. Allow to sit at room temperature for 15 minutes. Pipet up and down frequently. If sample is very concentrated, allow to sit at 4C overnight.
To quantitate, OD 1ul in 500ul water. Remember to add 1ul of formamide to the blank as well to account for any background from the formamide.
Should the sample be too dilute, it can easily be precipitated like a water-solubilized sample, and resuspended in a smaller volume with 3M Sodium Acetate and 100% ETOH.
After eluting the RNA from the columns with water, either dry down the sample, or precipitate with 3M Sodium Acetate and 100% ETOH.
Spin, resuspend in formamide, and OD
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Protocol by Tim Fitzwater
100x TAE buffer 5 blotting buffer for Northerns
[1x] | [100x] | |||
10 mM | 1 M | 121.14 g | Tris?base | |
0.5 mM | 50 mM | 18.61 g | Na2EDTA | |
5 mM | 500 mM | 41.04 g | NaOAc-anhydrous | |
~43 mL | Glacial acetic acid to pH 7.8 | |||
x mL | Type I water | |||
1000 mL |
100x TAE5 is composed of 1 M Tris base, 50 mM Na2EDTA, 500 mM NaOAc and approximately 43 mL of glacial acetic acid (to pH 7.8) per liter of 100x buffer.
Autoclave or filter sterilize. This buffer is used with the Hoeffer/LKB apparatus to transfer RNA to nitrocellulose or nylon. The Hoeffer/LKB box requires almost 5 liters of 1x TAE5. Gels do not need to be presoaked to remove urea. See the Northern protocol.
Acetate is oxidized to carbonate during electrophoresis, raising pH.
50x Denhardt誷
1% | 5 g | Ficoll | |
1% | 5 g | polyvinylpyrolidone | |
1% | 5 g | acetylated BSA | |
x mL | Type I water | ||
500 mL |
Dissolve with mild heat and stir. Spin 2000 rpm for 15 min. Filter sterilize supernatant through 0.45 ?unit with 2 prefilters. 50x stock is difficult to filter sterilize unless prefilters are used. Store aliquots at -20. Do not thaw at elevated temperatures.
20x SSPE
175.3 g | NaCl | ||
27.6 g | NaH2PO4 | ||
7.4 g | Na2EDTA | ||
800 mL | Type I water | ||
?/span>20.5 mL | 10 N NaOH to pH 7.4 | ||
x mL | Type I water | ||
1000 mL |
Autoclave or filter sterilize. SSPE can replace SSC plus NaPO4, pH 6.7.
50% Dextran sulfate MW 5000
2 g | Dextran sulfate MW 5000 | ||
x mL | Type I water | ||
4 mL |
Dextran sulfate MW 5000 works just as well as the traditional MW 500,000 material, but is significantly easier to dissolve. Weigh dextran sulfate directly into anti-static treated tube (do not try to transfer from weighing paper or weigh boat).
Dextran sulfate supports the growth of bacteria. Store at -20.
1. A Hoeffer/LKB Transphor unit is recommended for Northern blots. The Bio-Rad unit has been known to melt when incorrect voltage was applied.
The recommended transfer membrane for very small RNA was 0.1 祄 nylon (Schleicher & Schuell Nytran or ICN Biotrans) but this material is no longer available. RNA that is <110 nucleotides will tend to go through a 0.2 祄 membrane. Try Schleicher & Schuell nitrocellulose membranes (BA79/0.1 祄 and BA75/0.05 祄)? Nylon (neutral or charged) membranes are rated for denatured > 50 bp DNA fragments, require alkaline transfer conditions and may have higher backgrounds.
Hoeffer sponges are 6?x 9?and the gel must be cut to fit these dimensions.
