13. Surface RNase decontaminant solution.
14. TRIzol™ reagent or other commercially available guanidinium
thiocyanate-acidic phenol solution (see Note 3).
15. Chloroform.
16. Isopropanol.
17. 75% ethanol in RNase-free water (see Note 4).
18. RNase-free H 2 O.
19. DNase treatment kit (see Note 5).
20. UV spectrophotometer.
21. Tris-acetate-EDTA (TAE) running buffer (50Â): 242 g of Tris
base, 57.1 ml of glacial acetic acid, 100 ml of 0.5 M EDTA,
pH 8.0. Adjust the volume to 1 L with distilled water (the pH
should be around 8.5). Dilute the solution with ultrapure
water to 1Â for use.
22. Nucleic acid staining such as ethidium bromide (supplied in a
dropper bottle at 625 μg/ml).
23. 1% agarose: 1 g of agarose in 100 ml of TAE running buffer.
Add one drop (about 25 μg/40 μl) of ethidium bromide in
50 ml of the solution before agarose polymerization.
24. 6Â gel loading buffer for nucleic acid: 10 mM Tris–HCl,
pH 7.6, 60% glycerol, 60 mM EDTA, 0.03% bromophenol
blue, 0.03% xylene cyanol FF. Mix 1 volume of the 6Â gel
loading buffer with 5 volume of RNA solution (containing
0.5–1 μg of RNA).
25. Gel equipment for nucleic acid electrophoresis.
26. Electrophoresis power supply.
27. UV transilluminator to visualize nucleic acids.
3 Methods
Great care should be taken to prevent RNA degradation by exogenous RNases. Gloves should be worn at all times and changed
frequently. People with long hair should secure it. If possible, a
designated laboratory space should be reserved exclusively for RNA
extraction and manipulation (see Note 6). All the consumable
materials (tips, tubes) and solutions should be RNase-free and
protected from the dust. All the non-disposable materials that will
be in contact with the RNA (pipettes, benches, centrifuge, gel
equipment) should be washed with a surface RNase decontaminant
solution (see Note 7). All the steps are performed at room temperature unless otherwise noted.
44
Crispin Zavala-Alvarado and Nadia Benaroudj
14. TRIzol™ reagent or other commercially available guanidinium
thiocyanate-acidic phenol solution (see Note 3).
15. Chloroform.
16. Isopropanol.
17. 75% ethanol in RNase-free water (see Note 4).
18. RNase-free H 2 O.
19. DNase treatment kit (see Note 5).
20. UV spectrophotometer.
21. Tris-acetate-EDTA (TAE) running buffer (50Â): 242 g of Tris
base, 57.1 ml of glacial acetic acid, 100 ml of 0.5 M EDTA,
pH 8.0. Adjust the volume to 1 L with distilled water (the pH
should be around 8.5). Dilute the solution with ultrapure
water to 1Â for use.
22. Nucleic acid staining such as ethidium bromide (supplied in a
dropper bottle at 625 μg/ml).
23. 1% agarose: 1 g of agarose in 100 ml of TAE running buffer.
Add one drop (about 25 μg/40 μl) of ethidium bromide in
50 ml of the solution before agarose polymerization.
24. 6Â gel loading buffer for nucleic acid: 10 mM Tris–HCl,
pH 7.6, 60% glycerol, 60 mM EDTA, 0.03% bromophenol
blue, 0.03% xylene cyanol FF. Mix 1 volume of the 6Â gel
loading buffer with 5 volume of RNA solution (containing
0.5–1 μg of RNA).
25. Gel equipment for nucleic acid electrophoresis.
26. Electrophoresis power supply.
27. UV transilluminator to visualize nucleic acids.
3 Methods
Great care should be taken to prevent RNA degradation by exogenous RNases. Gloves should be worn at all times and changed
frequently. People with long hair should secure it. If possible, a
designated laboratory space should be reserved exclusively for RNA
extraction and manipulation (see Note 6). All the consumable
materials (tips, tubes) and solutions should be RNase-free and
protected from the dust. All the non-disposable materials that will
be in contact with the RNA (pipettes, benches, centrifuge, gel
equipment) should be washed with a surface RNase decontaminant
solution (see Note 7). All the steps are performed at room temperature unless otherwise noted.
44
Crispin Zavala-Alvarado and Nadia Benaroudj