DNA Extraction Protocols Using Silica

Below we present the protocols we have used to isolate DNA from various tissues using Silica and Guanidinium Thiocyanate.These protocols are adapted from Boom et al. (1990),Höss & Pääbo (1993),and Höss (1994).We highly recommend reading these references before using these protocols. We thank M. Höss and A. Cooper for providing helpful and unpublished information.

Additional variations on this theme have been developed. Carter & Milton (1993) present a simpler protocol.Höss et al. (1992) combined prot.K digestions with silica extraction to get DNA from bear poop.Gerloff et al. (1995) combined the methods of Carter & Milton with the Boom et al.(1990) and Höss & Pääbo (1993) to extract DNA from bonobo poop. Constable et al. (1995) combined silica methods with CTAB and phenol extractions to get DNA from baboon poop. So,as you can see,there are lots of variations that could be done.Note also that much of this extraction from poop has made its way into Nature.

Note: Carry out all procedures in a fume hood using PCR-free chemicals and supplies.

Materials needed (suggested stocks are sufficient for several hundred "extractions"): Silica - ~100 g, Sigma # S-5631 GuSCN - ~500 g, Amresco (cheaper) # 0380 or Sigma # G-6639 Triton X-100 - ~10 mL 0.2 M EDTA (pH 8.0) - ~100 mL Tris-HCl (pH 6.4) - see Höss (1994), ~250 mL Ethanol (95% or 70%) - 500 mL NaCl 50 mL Conical Centrifuge Tubes - 1.5 mL centrifuge tubes Aluminum Foil 10M HCl

I. Preparation of solutions

A. Silica - note: 2 day procedure

1. Add 6 g SiO2 to a 50 mL Falcon tube, and fill to the 50 mL mark with deionized water (dH2O).

2. Vortex and allow to settle 24 h at room temperature.

3. Remove the upper 43 mL of liquid (aspirate).

4. Refill to 50 mL with dH2O, vortex and allow to settle 5 h at room temperature.

5. Remove the upper 44 mL of liquid.

6. Add 60 µL 10M HCl.

7. Vortex (homogenize) and aliquot for storage. Höss recommends 500 µL aliquots wrapped in aluminum foil (stable for 6 months at room temperature).

B. Extraction buffer

1. Combine 24 g GuSCN (guanidinium thiocyanate) and 20 mL 0.1 M Tris-HCl (pH 6.4) in a 50 mL Falcon tube.

2. Heat to 60 oC to dissolve the GuSCN.

3. Add 4.4 mL of 0.2 M EDTA (pH 8.0).

4. Add 0.5 mL Triton X-100.

5. Mix by inverting.

6. Add 0.5 mL of silica suspension (see above), mix, and let sit for at least 1 h at room temperature (mixing sporadically).

7. Spin out the silica by centrifugation. Save the supernatant wrapped in aluminum foil or transfer to a brown bottle (stable 1 month at room temperature).

Note: Steps 6 & 7 remove contaminating DNA from the buffer.

C. Washing buffer - note: make double this amount to equal extraction buffer use per sample

1. Combine 24 g GuSCN (guanidinium thiocyanate) and 20 mL 0.1 M Tris-HCl (pH 6.4) in a 50 mL Falcon tube.

2. Heat to 60 oC to dissolve the GuSCN.

3. Add 4.4 mL of 0.2 M EDTA (pH 8.0).

4. Mix by inverting.

5. Add 0.5 mL of silica suspension (see above), mix, and let sit for at least 1 h at room temperature (mixing sporadically).

6. Spin out the silica by centrifugation. Save the supernatant wrapped in aluminum foil or transfer to a brown bottle (stable 1 month at room temperature).

D. Washing Ethanol

70% EtOH supplemented with 10 mM NaCl (e.g., for 500 mL add: 368.4 mL 95% EtOH, 1.25 mL 4M NaCl, fill to 500 mL with dH2O).

II. Procedures

Below we present several protocols that are useful when extracting DNA from various tissues. Several parameters can be altered, however, one should keep in mind: 1) it is very difficult to break-up silica pellets when more than 100 µL of silica is used, 2) the total amount of DNA recovered depends not only on how much is initially bound to the silica, but also how well it is eluted off in TE, and 3) if the guanidine thiocyanate becomes acidic, hydrogen cyanide will be formed, thus it is an extremely good idea to do all manipulations in a fume hood!

Set-up:

in the hood: 3 - 500+ mL side-arm flasks, one attached to vacuum line, ready to be used for aspiration (see Sambrook et al. 1989:1.27), a vortexer, 1 mL pippeter & tips, solutions prepared above,

in or near the hood: a microfuge.

A. General Protocol for “Soft” Tissues (cf. Boom et al. 1990)

1. Combine 900 µL extraction buffer and 40 µL silica suspension to a 1.5 mL microfuge tube and vortex.

2. Add tissue and vortex.

3. Let sit 10 minutes at room temperature.

4. Vortex briefly to break apart the silica, then centrifuge (15 s) at 12,000 x g. Discard supernatant by aspirating into the side-arm flask.

5. Wash the silica pellet twice with washing buffer by adding1 mL buffer, vortexing (in some cases it is faster to use a pipette tip to break apart the pellet), & centrifuging (15 s at 12,000 x g). Discard (aspirate) the supernatant each time.

6. Wash twice with 70% ethanol as above (add 1 mL, vortex, centrifuge). Discard (aspirate) supernatants into the second side-arm flask.

7. Dry the final pellet in the fume hood with the cap open or in the speed-vac using low heat.

8. Resuspend the pellet in 75 µL TE (10 mM Tris-HCl, 1 mM EDTA).

9. Vortex briefly and incubate at least 10 minutes (up to several hours) at 55oC.

10. Vortex. Centrifuge 2 minutes at 12,000 x g. Transfer the supernatant to a new tube.

11. Repeat steps 8-10, combining the second aliquot of TE with the first in step 10. If significant amount of silica are carried into the new tube, then it may be worthwhile to spin again & transfer the supernatant into another new tube (see note below).

12. Store at 4oC (short term - days to weeks) or -20oC (long term).

Note: ensure that there is no residualsilica before using DNA from these protocols. If there is residual silica, then we recommend one of the following: 1) vortex the tubes briefly, centrifuge for 1-2 min. at 12,000 x g, then take out supernatant without disturbing the silica on the bottomof the tube; or 2) vortex the tubes briefly, centrifuge for 1-2 min. at 12,000xg, then transfer the supernatant to a new tube.

B. Protocol for Non-mammalian Vertebrate Blood.

Note: This protocol is designed for maximum yield obtainable in 1.5 mL tubes. Simply increasing blood or silica has resulted in lower yields in our hands.

1. Combine 900 µL extraction buffer and 75 µL silica suspension to a 1.5 mL microfuge tube and vortex.

2. Add 15 µL of blood and vortex.

3. Let sit 10 minutes at room temperature or elevated temperature (up to 65oC).

4. Proceed as above (follow the general protocol), except, elute DNA from the silica pellet by using 125 µL of TE, twice.

C. Protocol for “Hard”Tissues (cf. Höss & Pääbo 1993).

1. 500 mg tissue is combined with 1 mL extraction buffer in a 1.5 ml microfuge tube.

2. Incubate at 60oC for at least 1 h with sporadic agitation.

3. Spin down debris (1 min. at 12,000 x g) and transfer 500 µL of the supernatant to a fresh microfuge tube.

4. Add 500 µL extraction buffer and 40 µL silica suspension.

5. Proceed from step 3 of the general protocol.

Waste Disposal:

The aspirated extraction and washing buffers should be placed in a brown glass bottle with a minimum of 1/20th volume 10N NaOH. This bottle should be kept under the fume hood and should be disposed of as hazardous waste.

The aspirated 70% EtOH can usually be disposed of in the same manner as any alcohol waste.