Nucleated blood cells are used to prepare genomic DNA. The cells are first washed, then lysed and the nuclei (left intact) are pelleted in a low speed centrifuge run. The nuclei are resuspended in a small volume, and lysed with SDS and proteinase K. Phenol and chloroform extractions follow to remove most of the non-nucleic acid organic molecules. The DNA is precipitated out of solution with isopropanol, collected on a glass hook, and resuspended in TE. The A260, A280 spectrophotometer readings are taken, quantity of DNA present calculated, and theDNA reprecipitated in the presence of high concentrations of ammonium acetate (to further purify the DNA). The DNA is resuspended in TE to approximately 300 ug/ml , and another A260 and A280 spectrophotometer readings are taken to verify the concentration and DNA quality. The final concentration is adjusted to 250 ug/ml with TE.
Because the ABI nucleic acid extractor is very efficient in producing high-quality DNA preparations, we no longer depend on this manual procedure for routine extractions. However, it is the method of choice for larger preparations (e. g. preps of DNA from 500 ml blood used for screening blots), because the ABI extractor cannot handle the larger quantity of cells unless the sample is divided into many small aliquots (creating more work for the operator and defeating the purpose of using a machine to do the work). The method works equally well for preparing DNA from lymphoblastoid cell lines.
Caution: Human blood may contain infectious agents and should be treated as if it does. Always wear gloves and work in a hood until the DNA is ready for the first precipitation. Sterilize aqueous wastes by autoclaving after treatment with disinfectant. Use all plastic disposable materials to avoid contaminating the DNA with plasmid or phage DNAs, and use the pipetman set designated 'for human DNA use only' when aliquoting the DNA.
- Wash the white blood cells (the buffy coat from 500 ml blood, or approximately 30 ml WBC from a pheresis) twice with cold normal saline (0.9 % NaCl). Wash by resuspending cells in 20 ml of saline in a 50 ml orange-capped tube and spinning at 1500 rpm in the Beckman TJ-6 or J 21 for 15 minutes at 4deg.C. Pour off supernatant into a beaker containing disinfectant, and repeat wash.
- Add 20 ml of cold buffer A to the pellet, and vortex 2 minutes. (Cells are lysed without lysing nuclei.) Spin at 1500 rpm, 15 minutes, 4deg.C. Expect a pure white pellet about half the size of the cell pellet. Pour off the supernatant and wash the nuclei with 20 ml of buffer A.
- Add 10 ml of cold buffer B to the pelleted nuclei, and resuspend by vortexing. Add 1/10 volume (1 ml) of 10% SDS and swirl to mix. Add 1/100 volume (110 ul) of proteinase K (10 mg/ml) to a final concentration of 100 ug/ml. Mix gently. Incubate at 37deg.C overnight with gentle shaking on an orbital shaker or a rocker. The solution should be viscous. Treat the DNA solution gently from this point forward to reduce the amount of DNA shear.
- Add an equal volume of buffer-equilibrated phenol, tightly cap and parafilm the tube, and mix gently at room temperature for 4 hours (can be done overnight) on a rocker or orbital shaker. If the ABI phenol- chloroform is used instead of the buffer-equilibrated phenol, extract overnight, and follow with a second extraction for 2 hours the following morning.
- Separate the phases by spinning at 2500 rpm for 15 minutes in a Beckman low speed centrifuge. Save the aqueous (top) layer. Using a 10 ml plastic pipet, slowly pipet out the top layer and transfer to a clean 50 ml orange- capped tube.
- Add an equal volume of chloroform: isoamyl alcohol (24:1) and mix gently on a shaker at room temperature for 15 to 30 minutes. Separate the phases by centrifugation at 2500 rpm for 15 minutes.
- Transfer the aqueous (top) phase to a clean orange-capped tube. Add 1/10 volume (1.1 ml) of 3M sodium acetate. Add 1.5 volumes (18 ml) isopropanol. Cap the tube tightly and invert tube repeatedly until a tight precipitate forms.
- Remove the precipitate by capturing it with a hooked Pasteur pipet. (Flame seal the tip of a short pasteur pipet, then heat the end to form a small hook. Store hooks in ethanol until use.) Drain all excess liquid from the precipitate by sliding it gently against the side of the tube and/or touching it lightly with the edge of a kimwipe. Place the precipitate in 10 ml of TE in a clean 50 ml orange-capped tube. Place on a rocker or orbital shaker at 4deg.C until the DNA is completely dissolved ( at least 24 hours, sometimes 3- 4 days). If DNA does not appear to be uniformly dissolved after two days, add more TE and continue mixing.
Days 3 to 7
- After the DNA is completely resuspended, make a 1/25 to 1/50 dilution in TE, and take spectrophotometer readings of A260 and A 280. If the actual A260 reading taken is higher than 0.500 (the point where the readings start to be inaccurate), increase the dilution factor and repeat the spectrophotometer readings. The A260/280 ratio should be close to 1.9 (deviations of more than + or - 0.2 indicate presence of impurtities which may later interfere with enzyme activity). Calculate the DNA concentration as follows:
A260 x dilution factor x 50 (DNA at 50 ug/ml has an A260 of 1) = ug/ml DNA.
Find the total yield (in ug) by multiplying the concentration by the total volume (in mls).
- Reprecipitate the DNA by adding 1/2 volume of 7.5 ammonium acetate and 2 volumes of cold 95-100% ethanol. Collect the precipitate as before and rinse it in 1 ml 70 % ethanol in an eppendorf tube. Repeat the rinse with 100 % ethanol, drain as before and redissolve in the appropriate amount of TE to obtain a final concentration of 300 ug/ml.
- After the DNA is completely resuspended, make a 1/25 dilution in TE and take spectrophotometer readings of A260 and A 280. Adjust the concentration of the DNA to 250 ug/ml with TE. (The acceptable concentration range needing no further adjustments is 225-275 ug/ml.) If the concentration is too low, reprecipitate with 1/10 vol sodium acetate, and assume a 10 % loss of DNA when figuring the amount of TE needed for resuspension.
- Prepare several 1 ml aliquots for working solutions and store these and the remaining DNA solution at 4deg.C. The yield is between 2 and 6 mg per 8 x 10e8 white blood cells.
Procedure for DNA extraction from whole blood:
- Take the blood sample in standard heparinized vacutainer tubes (Becton-Dickinson). The sample may be stored at least 3 days in the refrigerator or shipped on ice.
- Add 9 volumes of buffer A to the whole blood. Mix well and hold on ice 2 minutes. Spin at 1500 rpm in the Beckman low speed centrifugeat 4deg.C for 15 minutes. Expect a pink pellet of nuclei.
- Continue with the normal procedure (step 3 above) if the sample was at least 30 ml blood, but adjust the concentrations accordingly for smaller samples (the DNA will not form visible precipitate if too dilute). After isopropanol precipitation, redissolve the DNA in 1 ml TE. Expect yields of 0.2-1 mg DNA from 20-30 ml of whole blood.
0.32 M sucrose 109.5 g sucrose 10 mM Tris HCl pH 7.6 10 ml 1 M Tris-HCl pH 7.6 5 mM MgCl2 5 ml 1M MgCl2 1 % Triton-X-100 bring volume to 1 liter with deionized water sterilize above solution by autoclaving, then add 10 ml Triton-X-100 Buffer B
25 mM EDTA pH 8.0 50 ml EDTA pH 8.0 75 mM NaCl 40 ml 5 M NaCl bring volume to 1 liter with deionized water sterilize by autoclaving
Normal saline (0.9 % NaCl)
9 g NaCl Ammonium Acetate, 7.5 M
bring volume to 1 liter with deionized water sterilize by autoclaving
578.1 g NH4 Acetate
bring volume to 1 liter with deionized water filter sterilize
Prepare fresh in sterile water to 10 mg/ml
Chloroform: isoamyl alcohol (24:1)
Prepare in the chemical fume hood: measure 240 ml chloroform using a 250 ml glass cylinder add 10 ml isoamyl alcohol mix by pouring into a sterile bottle. Store in the hood. Phenol, saturated solution (neutral buffer equilibrated)
- Dissolve 500 g phenol in an approximately equal volume of 50 mM Tris-HCl, pH 8.0. (Heating to 60 deg.C may be necessary to achieve rapid dissolution. AnalaR phenol dissolves readily at room temperature.)
- To the dissolved phenol at room temperature, add 33 ml of 2.5 M Trizma base (30.3 g Trizma / 100 ml). Mix well and decant the upper phase after allowing the phases to separate completely.
- Add an equal volume of 50 mM Tris-HCl, pH 8.0, mix and decant upper phase as above.
- Repeat step 3.
- Add about 1/5 volume 50 mM Tris-HCl, pH 8.0 to cover the phenol and store in a brown or foil- covered container at 4deg.C.
Note: phenol used for human DNA isolation should be labeled "For human DNA only!"
CRI Laboratory Manual: RFLPs Project (1989).