1、Rapid Extraction of High Quality DNA from Whole Blood Stored at 4C for Long PeriodMaterials and MethodsStandard chemicals This method uses standard chemicals that can be obtained from any major supplier; we used chemicals supplied by Sigma Co. as follow: EDTA (0.5 M), pH 8.0: Add 186.1 gr of anhydro
2、us EDTA to 800 ml of distilled water. Adjust pH to 8.0 with NaOH pellets. Make up to 1 liter with distilled water. Autoclave at 15 p.s.i. for 15 min. 1 M Tris-HC1, pH 7.6: Dissolve 121.1 gr of Tris base in 800 ml of distilled water. Adjust pH with concentrated HCl. Allow mixture to cool to room temp
3、erature before finally correcting pH. Make up to 1 liter with distilled water. Autoclave at 15 p.s.i. for 15 min. Preparation of Red blood cell lysis buffer: 0.01 M Tris-HCl pH 7.6, 320 mM sucrose, 5 mM MgC12, 1% Triton X 100. Add 10 ml of 1 M Tris, 109.54 gr of sucrose, 1.01 gr MgC12, adjust pH to
4、8.0 and finally add 10 ml of Triton X-100 to 800 ml of distilled water, and make up to 1 liter with distilled water. Autoclave at 15 p.s.i. for 10 min. Sugars at high temperature can cause caramelization (browning), which degrades the sugars 5. Preparation of Nucleic lysis buffer: 0.01 M Tris-HC1, 1
5、1.4 mM sodium citrate, 1 mM EDTA, 1 % sodium dodecyl sulphate (SDS). Take 10 ml of 1 M Tris-HC1 (pH 7.6), 3.75 gr of anhydrous EDTA (pH 8.0), 10 gr SDS, 2.94 gr of sodium citrate, and adjust pH to 8.0. Make up to 1 liter with distilled water. Autoclave 15 min at 15 p.s.i. TE Buffer, pH 8.0: Take 5 m
6、l of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA, pH 8, and make up to 1 liter with distilled water. Adjust pH to 8.0 and autoclave 15 min at 15. p.s.i. Chloroform prechilled to 4C. Ethanol (100%) prechilled to -20C. Procedure of DNA Extraction Before starting DNA extraction, liquid blood venogects sho
7、uld be shake gently by rotating blood mixer (vortex) 1. Pour 500 l of blood into a 1.5 ml eppendorf tube and add 1000 l of red cell lysis buffer. 2. Shake microfuge tube gently (up to homogenizing), then spin for 2 minutes at 7000 rpm. 3. Discard supernatant and repeat steps 1-3 two or three more ti
8、mes to remove hemoglobin. It is important to breakdown the pellet by vortexing and rinses it well in red blood cell lysis buffer in order to clean the white blood cells from residual of hemoglobin. 4. Placing the tube on tissue paper for few seconds downward. Be careful from cross-contamination betw
9、een different samples. 5. Add 400 l of nucleic lysis buffer to eppendorf tube. Note: if the pellet formed, you must pipette the pellet up to dissolve it. 6. Add 100 l of saturated NaCl (5M) and 600 l of chloroform to eppendorf tube and mix on a rotating blood mixer at room temperature then spin it f
10、or 2 minutes at 7000 rpm. 7. Transfer 400 l of supernatant to a new 1.5 ml tube. 8. Add 800 l of cold (-20C) absolute Ethanol and shake it gently then vortex it. DNA should appear as a mucus-like strand in the solution phase. 9. Spin the microfuge tube for one minute at 12000 rpm to precipitate, the
11、n discard supernatant carefully and let tube be completely dried in room temperature (Place Eppendorf tube downward on the tissue paper). 10. Add 50l of TE to it then vortex; keep eppendorf tube of DNA in 4C or -20C for later uses. We routinely use about one l per PCR reaction without adverse affects. DNA can be quantified and diluted to a working concentration at this point or simply use 1 l per PCR reaction. We expect that the yield of this procedure be 100 to 300 ng/l, DNA. Using the above method, high quality DNA samples from a sheep population were extracted for gene mapping studies.
