The method mentioned here is suitable for a lazy person like me, because lysates can be left for a long time at room temperature. It is also a good method, because high molecular weight DNA can be easily extracted killing inherent nucleases.
Step-by-Step Protocol
Dispense 400uL of TNESU8 buffer into an Eppendorf 1.5mL tube.
Put 100-300mg of muscle tissue etc. Mincing is unnecessary but it is better to tear into a few pieces. In case of whole blood, centrifuge the blood cells to remove serum, which contains much proteins, before TNESU8 treatment.
Shake vigorously.
Leave it for several hours to several months (up to three years).
Add 20uL of PK solution.
Invert several times to mix.
Lysis for at least one month at room temperature, or several days at 37oC. Check fluidity of the lysate inverting the tube. After the lysate becomes enough fluid, proceed to the next step.
Add 50uL of 5M NaCl
Invert several times to mix.
If it is difficult to mix, prolong the lysis some more.
Add 500uL of Phe-Chl.
Gently rotate for 5-15min. If much debris would appear, prolong this step.
Centrifuge 5 min at 15000xg.
Take sup to a new tube.
Add 1000uL of ice-cold EtOH.
Gently invert several times. DNA fibers precipitate, and then mix well up to the viscous lysate becomes completely invisible. Do not freeze, or urea precipitates.
Microfuge for 10 sec. Do not use a high-speed centrifuge, unless the DNA were invisible or degraded. Non-DNA debris precipitates upon high-speed centrifugation.
Discard sup.
Wash the DNA pellet twice with 70% EtOH.
Add 200uL of TE buffer containing RNase.
Dissolve DNA in a refrigerator. Complete dissolution is important. Tap or invert the tube to dissolve the DNA. If the DNA pellet were stubborn to dissolve, click here.
Repeat NaCl, Phe-Chl, EtOH steps in the smaller scale.
Dry the DNA pellet in a clean-air chamber.
Dissolve in an appropriate volume of TE.
REFERENCE
Asahida T, Kobayashi T, Saitoh K, Nakayama I. 1996. Tissue
preservation and total DNA extraction from fish stored at ambient temperature
using buffers containing high concentration of urea. Fish Sci 62:727-730.
Buffers and Reagents
TNESU8 buffer
Tris-HCl pH7.5 10-50mM
NaCl
125mM
EDTA-3Na
10mM
SDS
1%
Urea
8M
Autoclaved and stored at room temperature. Stable for more than one year.
PK
5M NaCl
Phe-Chl (phenol-chloroform)
RNase stock solution
2M Tris-HCl (pH7.5 or 8) (for 1000mL)
Proteinase-K powder
100mg
50% glycerol (autoclaved) 5mL
Mix well, aliquot, and stored at -20oC.
Stable for more than one year. Do not weigh the powdered proteinase-K.
Instead, buy always pre-weighed reagent.
NaCl
292.2g
Add milli-Q water up to 1000mL.
Autoclaved and stored at room temperature. Stable for more than one year.
Prepare tris-saturated phenol and mix it with equal volume of chloroform.
To prepare the tris-saturated phenotl;
Phenol crystal
100g
Add 0.1g of 8-quinolinol.
Dip the bottle in a water bath at 65-70
Add 50mL of 2M Tris-HCl (pH8) and 50mL of milli-Q water (100mL of 1M Tris-HCl). Mix well. The phenol absorbs water and swells.
Discard sup.
Add 200mL (or as much as possible if it is in a small bottle) of 0.1M Tris-HCl (pH8). Mix well. Discard sup.
Repeat this step.
Add some Tris-HCl (0.1M pH8) to cover the tris-saturated phenol to avoid contact with air. Stored for several months in a dark refrigerator.
Buy pre-weighed (100g) phenol in a glass bottle (larger bottle is better) and process it in the bottle. Do not handle crystal phenol outside. Crystal phenol is stable for more than one year in a dark freezer.
To prepare Phe-Chl;
Take a small amount of tris-saturated phenol from the stock. Add equal volume of chloroform. It is convenient to prepare it in a 50mL falcon tube.
Stable for several months under dark refrigeration. Freshly prepared Phe-Chl is light yellow. It gradually turns to dark orange upon degradation.
RNase-A powder
100mg
TE buffer
10mL
Mix well, aliquot to Eppendorf 1.5mL tubes. Lock the cap and boil for about 5 min. Slowly cool down to room temperature, and denatured RNase anneals to be reactivated. Stored at -20
Tris
242.2g
Add milli-Q water up to about 800mL. Stir to dissolve.
Adjust pH with conc HCl. Because much (about 100mL or more) HCl is necessary, do not add too much (more than 800mL) water at the beginning.
After the pH adjustment, add milli-Q water up to 1000mL.
Autoclaved and stored under refrigeration. Stable for more than one year.
