RNA Later
RNAlater
0.5 M EDTA disodium, dihydrate (18.61 g/100 ml, pH to 8.0 with NaOH while stirring);
1 M Sodium Citrate trisodium salt, dihydrate (29.4 g/100 ml, stir to dissolve);
Ammonium Sulfate, powdered;
Sterile water.
In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1 M Sodium Citrate, 700 gm Ammonium Sulfate and 935 ml of sterile distilled water, stir on a hot plate stirrer on low heat until the Ammonium Sulfate is completely dissolved. Allow to cool, adjust the pH of the solution to pH 5.2 using 1 M H2SO4. Transfer to a screw top bottle and store either at room temperature or refrigerated.
In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1 M Sodium Citrate, 700 gm Ammonium Sulfate and 935 ml of sterile distilled water, stir on a hot plate stirrer on low heat until the Ammonium Sulfate is completely dissolved. Allow to cool, adjust the pH of the solution to pH 5.2 using 1 M H2SO4. Transfer to a screw top bottle and store either at room temperature or refrigerated.
Final concentrations:
25 mM Sodium Citrate
10 mM EDTA
70 g ammonium sulfate/100 ml solution
pH 5.2
0.5 M EDTA
Protocol 2
Add 186.1 g EDTA disodium salt dihydrate (Mallinckrodt 4931) to 700 ml MilliQ water
Stir to dissolve
Bring pH to 8.0 with NaOH (~50ml of 10M NaOH or ~23g NaOH pellets)
Bring volume to 1L with MilliQ water
Autoclave Prepare 1 M Sodium citrate
Add 294 g Na Citrate dihydrate (Mallinckrodt 0754-12) to 700ml MilliQ water
Stir to dissolve
Bring volume to 1L with MillQ water
Autoclave In a 2 or 3 L flask mix 1,400 mL MilliQ water with 60 mL 0.5M EDTA and 37.5 mL 1M sodium citrate with stirring
While continuing to stir the solution, add 1.05 kg (= 1,050 g) Ammonium sulfate in 100 g amounts
Stir ~30 min on hot plate set to low
it is possible that a tiny amount ammonium sulfate will not go into solution. This is okay; the most important thing is to have a saturated ammonium sulfate solution. Crystals should dissolve in ~30min
Allow solution to cool at room temperature for at least 30min, but overnight on bench is best
Filter solution through 0.2 μm filter to remove particles
Store at room temp or at 4°C
Add 186.1 g EDTA disodium salt dihydrate (Mallinckrodt 4931) to 700 ml MilliQ water
Stir to dissolve
Bring pH to 8.0 with NaOH (~50ml of 10M NaOH or ~23g NaOH pellets)
Bring volume to 1L with MilliQ water
Autoclave Prepare 1 M Sodium citrate
Add 294 g Na Citrate dihydrate (Mallinckrodt 0754-12) to 700ml MilliQ water
Stir to dissolve
Bring volume to 1L with MillQ water
Autoclave In a 2 or 3 L flask mix 1,400 mL MilliQ water with 60 mL 0.5M EDTA and 37.5 mL 1M sodium citrate with stirring
While continuing to stir the solution, add 1.05 kg (= 1,050 g) Ammonium sulfate in 100 g amounts
Stir ~30 min on hot plate set to low
it is possible that a tiny amount ammonium sulfate will not go into solution. This is okay; the most important thing is to have a saturated ammonium sulfate solution. Crystals should dissolve in ~30min
Allow solution to cool at room temperature for at least 30min, but overnight on bench is best
Filter solution through 0.2 μm filter to remove particles
Store at room temp or at 4°C
Note: If you read the patent application, they refer to an ammonium sulfate concentration of 700g per L. What they really mean is add 700g to 1L (including EDTA and sodium citrate at conc. listed above), so you end up with more than 1L total volume.
Note: The pH should be ~7 using this recipe. The patent states that pH can range from 4-8; the preferred commercial pH is 5.2; I use pH 7 for bacterioplankton samples, but pH can be lowered by adding H2SO4.
Note: Since this is a storage solution for RNA, use high quality reagents ("molecular biology" grade) when preparing the buffer.
RNAprotect Bacteria Reagent:
- 4% (w/v) Tetradecyl-trimethyl-ammonium bromide (TTAB)
- 200 mM tartaric acid
- NaOH to pH 4
RNAprotect Cell Reagent:
- 4% (w/v) Tetradecyl-trimethyl-ammonium oxalate (TTAO)
- pH 6-7