Protocol:DNA from blood: phenol-chloroform method

DNA Preparation
The white blood cells (WBC) of peripheral blood are usually the most convenient source of human genomic DNA for DNA analysis with respect to haemoglobinopathies. It is estimated that 10 ml of whole blood yield approximately 250 &mu;g of DNA, more than sufficient for complete analysis of globin genes with the methods that are currently available (ie based on PCR). DNA is an extremely stable molecule, but enzymes which catalyse the breakdown of nucleic acids (nucleases) are found in all cells. In intact cells the DNA is found in the nucleus and thus is protected from the action of nucleases which are abundant in the lysosomes in the cytoplasm. However when cells are lysed, the membranes of the cell compartments are disrupted, allowing nucleases to come in to contact with the DNA. Thus the first stages of DNA extraction uses buffers which contain inhibitors of nuclease activity. Additionally all steps must be carried out at low temperatures (0oC). For long-term storage of samples prior to extraction a temperature of -70oC is recommended. There are many different methods described for DNA extraction from whole blood; the methods described below of salting–out and phenyl chloroform are used in the laboratories of some of the authors. There are also kits on the market for extracting DNA from blood samples which work well, but tend be expensive if used to prepare DNA from 5-10 ml blood samples.

DNA from blood:phenol-chloroform method
To prepare high-quality DNA the blood should be as fresh as possible. The handling of blood samples should be carried out with great care, and they should all be treated as potential sources of infection. As with all laboratory protocols the hazards of storage, handling and disposal of the various reagents must be assessed and addressed properly in the local laboratory standard operating procedure.

Reagents

 * 1) Reticulocyte saline: 5 mM KCl, 0.13 M NaCl, 7.4 mM MgCl2
 * 2) 10x Lysis mix: 0.77 M NH4Cl, 46 mM KHCO3. Dilute to 2x, to make red blood cell lysing mix.
 * 3) WBC Lysing solution: 100 mM NaCl, 25 mM EDTA
 * 4) 10% SDS [Sodium dodecyl sulphate] (make from 20% stock solution)
 * 5) 10 mg/ml proteinase K [ICN Pharmacueticals}
 * 6) Phenol (saturated with 0.1 M Tris) [Rathburn Chemicals]
 * 7) Chloroform
 * 8) 7.5 M Ammonium acetate
 * 9) 100% Ethanol (store in ‘flammables cabinet’ with lock)
 * 10) 70% Ethanol

Method

 * 1) Transfer 2-10 ml blood in EDTA to a 15ml tube. Spin at 3000 rpm for 10 min.
 * 2) Carefully remove the supernatant (plasma).
 * 3) Fill the tube to 15-30 mls with 2x lysis mix (dependent on quantity of blood material being lysed). Shake well and rotate for 10 min to lyse the red blood cells (the lysis will be complete when the solution is a clearish red).
 * 4) Spin at 3000 rpm for 10 mins. A pellet of white cells should form at the bottom of the tube. Carefully tip off the supernatant to waste leaving the pellet stuck to the bottom of the tube. Repeat steps 3 and 4 to get a cleaner/whiter/purer pellet.
 * 5) Add 450 &mu;l WBC lysing solution to an eppendorf tube labelled with the lab number. Add 30 &mu;l of 10% SDS. Transfer about half of the white pellet (dependent on quantity) to the 450 &mu;l lysing solution and SDS in the labelled eppendorf tube. Then transfer the rest of the white blood cell pellet to a new eppendorf together with 200 &mu;l of lysing solution, this is stored as Buffy Coats in the freezer as a back up. Add approximately 200 &mu;l of proteinase K (10 mg/ml) to the WBCs in lysing solution and SDS (dependent on quantity of WBCs). After pipetting up and down a few times the lysate should be viscous, it may be necessary to add more lysing solution at this stage.
 * 6) Incubate over night at 37oC or at 55oC for 4-5 hours, after which the solution should be less viscous, due to the white blood cells being broken down.
 * 7) Check that the sample has been lysed adequately before proceeding with the phenol-chloroform extraction. If the solution can move easily up and down a pastette then lysis is complete. If not add more proteinase K and incubate further.
 * 8) To the sample add an equal volume of phenol (saturated with 0.1 M TrisHCl). 700 &mu;l is usually added. Mix and spin in a microfuge at 3000 rpm for 5 min.
 * 9) Remove the upper aqueous layer to a clean appropriately labelled tube and add another equal volume of phenol.
 * 10) Remove the upper aqueous layer to a clean tube and add an equal volume of chloroform. Spin at 3000 rpm for 5 min.
 * 11) Remove the upper aqueous layer and add another equal volume of chloroform and spin again at 3000 rpm for 5mins.
 * 12) Remove the upper aqueous layer to a clean tube and add 1 &mu;l of 4 M Ammonium acetate. Add 2x volume of 100% ethanol and gently shake to precipitate the DNA.
 * 13) Spin at 3000 rpm for 5 min to pellet the DNA.
 * 14) Carefully pour off the 100% ethanol (taking care not to disturb the pelleted DNA) and add 500 &mu;l of 70% ethanol to wash the DNA. Shake well and spin at 3000 rpm for 5 min.
 * 15) Carefully tip off the 70% ethanol wash leaving the DNA stuck to the bottom of the tube. Air-dry the pellet until all the ethanol has evaporated off it should still be clear.
 * 16) Dissolve the pellet in an appropriate amount of sterile water.
 * 17) Leave at 4oC for 4-5 hours for the DNA to dissolve.
 * 18) The solvent waste from the phenol and chloroform spins should be dispensed into the solvent waste bin and disposed of according to local waste disposal rules. The tubes should then be disposed of in bins for autoclaving.