Standard Operating Procedure
Hazards:
*Phenol:
Extremely potent irritant of the eyes, mucous membranes, and skin; absorption
causes convulsions as well as liver, kidney, and other systemic damage.
*Chloroform: Avoid
Inhalation! Chloroform is a central nervous system depressant and carcinogen.
Inhalation of 10,000 ppm of chloroform vapor produces clinical anesthesia.
Inhalation of higher doses causes cardiovascular depression, with death
resulting from ventricular fibrillation. Containers of chloroform should be
protected from physical damage, heat sources, direct sunlight, and moisture,
and should be stored separately from acetone, alkalis, and chemically active
metals. Do not use rubber or plastic to transfer chloroform.
Protection:
If
phenol or chloroform contacts the skin, immediately wash affected areas with
soap and water.
Clothing
contaminated with phenol or chloroform should be removed immediately, and
laundered.
Waste:
Spill
Clean-Up:
In
the event of a spill or leak involving phenol, persons not wearing protective
equipment and clothing should be restricted from contaminated areas until
cleanup has been completed. For small liquid spills, take up with absorbent
material and place into closed containers for later disposal.
*
Procedure:
1.
For cells growing in suspension culture, or a single-cell suspension from
thymus or spleen:
Wash
cells in cold PBS 1X. Remove supn.
Gently
suspend cells in TE (pH 8.0) at or up to 5x10e7/ml
Add
10 ml of lysis buffer for each ml of cell suspension
Incubate
1 hour at 37oC
2. Transfer the lysate to a polypropylene
or glass tube that fits the Shimizu JA-17 rotor.
Add
proteinase K (20
mg/ml) to a final concentration of 100 ug/ml. Mix Gently
Incubate
3 hours at 50oC. Swirl from time
to time
3. Add an equal volume of phenol (pH 8.0). Mix well, but gently.
Centrifuge
5000xg (6500 rpm in JA-17) for 15 minutes at RT.
Transfer the aqueous phase (top) to a fresh centrifuge tube
using a Pasteur pipette.
4. Add an equal volume of a 1:1 mix of phenol:chloroform. Mix well, but gently
Centrifuge
5000xg (6500 rpm in JA-17) for 15 minutes at RT.
5. Transfer the aqueous phase(s) to a
fresh centrifuge tube (may need to split to 2 tubes here)
add 0.2 volumes of 10 M ammonium acetate
add 2 volumes of 100% ethanol at room temperature
Swirl until thoroughly mixed
Centrifuge 5000xg (6500 rpm in JA-17) for 5 minutes at RT.
6. Wash the DNA pellet twice with 70%
ethanol and collect by centrifugation as above.
7. Remove as much of the 70% ethanol as possible, using an
aspirator. Store the pellet
inverted at RT until visibly dry—about 30 minutes. (Do not dry
completely: dessicated DNA is very difficult to dissolve!)
8. Add 1ml of TE for every .1 ml of cells in Step
1. Gently rock overnight at 4oC to
dissolve. Store at 4oC.
***To
measure the concentration, draw a 10 ul sample with a cut-off yellow tip. Dilute in 500ul TE and vortex for 2
minutes. Measure the concentration
of this 1:50 dilution of the sample.
A good 260:280 ratio is 1.2, but this is not a reliable indicator of
purity.
Reagents:
Lysis
Buffer:
10 mM
Tris-Cl (pH 8.0)
0.1 M EDTA
(pH 8.0)
0.5% (w/v)
SDS
20ug/ml
DNase-free pancreatic RNAse ***add just before using
proteinase
K
Is
purchased as a powder. Resuspend
to 20 mg/ml in ddH2O and freeze in small aliquots.
Phenol
Must be pH 8.0. Usually sold as a solution pH 6.6, to which a vial of Tris buffer is added to bring to pH 8.0. There is a small amount of aqueous material at the top. Extend through this to pipette phenol. Use caution, since phenol is a strong toxic irritant.
Use glass
pipettes and glass or polypropylene tubes when using chloroform. It degrades
polystyrene and polycarbonate.
Avoid inhalation.
Volumes
worksheet:
Example:
5x10e7 cells _______
cells
TE 1ml _______
Lysis
Buffer 10ml _______
RNAse _______
Proteinase
K 55ul _______
Phenol 11ml
(total 22ml) _______
Phenol:Chlor 11ml (total 22 ml) _______
AmOAc 2.2ml
(total 13.2 ml) _______
Ethanol 26
ml (total 39 ml)(2 tubes) _______