John M. S. Bartlett.pdf - Bio-Nica.info

50 Stirling and Bartlett
4. Proteinase K: Prepare 1 mg/mL w/v DEPC water stock, which can be stored at –20°C
for up to 12 mo. Dilute in 10 mM Tris-Cl/0.5% SDS as required, and discard unused
diluted enzyme.
5. Phenol/chloroform/isoamyl alcohol: Phenol is presaturated with 10 mM Tris-HCl, pH 7.5.
Prepare a mixture of 25241 phenolchloroformisoamyl alcohol (v/v/v). Store at room
temperature for up to 6 mo, shielded from light.
6. TE Buffer, pH 7.6: take 10 mL of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA, and make
up to 1 L with distilled water. Adjust pH to 7.6. Autoclave 15 min at 15. p.s.i.
2.3. DNA Extraction (as for RNA Plus)
1. Dual extraction buffer: 0.1 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS.
Adjust to pH 12.0 with 5 N NaOH immediately before use.
2. 7.5 M ammonium acetate.
3. Methods
3.1. RNA Extraction
1. Tissues should ideally be collected fresh and stored in liquid nitrogen. Routinely samples
are collected on ice and transported for freezing within 30 to 60 min.
2. Tissues are disaggregated using a Braun-micro dismembranator. Teflon vessels and steel
ball bearings are cooled in liquid nitrogen before use. Frozen tissue (50 to 500 mg) is
placed in the vessel with a single ball bearing and agitated at 1000 cycles/second for 60 s.
The vessel is then recooled in liquid nitrogen. This process is repeated until tissue is
powdered (usually twice; see Note 3).
3. Immediately after disaggregation of tissue, material is resuspended while frozen in 1.5 mL of
LiCl/Urea and transferred to a separate tube. The vessel is washed a further 2× with 1.5 mL
of LiCl/Urea and the washing combined with the original sample. The resuspended medium
is made up to 6 mL in LiCl/Urea and sonicated for 2× 30 s at maximum power using a
probe sonicator. The sonicated samples are stored overnight at 4°C (see Note 4).
4. Centrifuge at 15,000g, 4°C for 30 min. The supernatant is discarded and the pellet washed
with a further 6 mL of lithium chloride/urea, recentrifuged (15,000g at 4°C for 30 min)
and the supernatant again discarded.
5. The pellet is resuspended in 6 mL of Tris-HCl/SDS with 50 µg/mL proteinase K (Boehringer
Mannheim, UK), and incubated at 37°C for 20 min.
6. Samples are mixed with an equal volume of phenolchloroformisoamyl-alcohol and
mixed by inversion several times.
7. After mixing, the sample is centrifuged at 2000g at room temperature for 10 min and
the aqueous phase recovered for RNA extraction, the organic phase is retained for DNA
extraction.
8. Repeat steps 6 and 7.
9. After the final extraction, 300 µL of 8 M LiCl and 2.5 volumes absolute alcohol are added
and samples stored at –20°C for 30 min overnight. RNA is pelleted by centrifugation at
4000g, 4°C for 45 min.
10. The supernatant is discarded and the RNA pelleted dried and resuspended in 50 µL of TE.
Concentrations are estimated by optical density at 260/280 nm.
3.2. DNA Extraction
1. Combine the organic phases, including the interfaces from step 7 above (both times), in
a 15-mL polypropylene tube.
2. Add an equal volume of extraction buffer, vortex for 1 min, and place on ice for 10 min.