How much volume of blood can i use? - the protocol is included (Sep/27/2007 )
Regarding this protocol:
Buffer A (Red blood cell lysis buffer) composition
0.32 M sucrose
10 mM Tris HCl
5 mM MgCl2
Adjust pH to 7.6
Buffer B (Proteinase K buffer) composition
20 mM Tris-HCl
4 mM Na2EDTA
100 mM NaCl
Adjust pH to 7.4
N.B. All solutions should be sterile. Buffer A should be autoclaved prior to addition of Triton-X-100. Sterile filtering of solutions instead of autoclaving is a better option.
Add 1 volume of buffer A to 1 volume of blood and 2 volumes of cold, sterile, distilled, deionised water. Vortex gently or invert tube 6-8 times and leave to incubate on ice for 2-3 minutes.
Spin at 3500 rpm for 15 minutes at 4oC. Discard supernatant into 2.5% bleach solution and re-suspend pellet (vortex for 30 seconds at medium speed) in 2 ml of buffer A and 6 ml of water. Spin at 3500 rpm for 15 minutes at 4oC. The pellet should be white to cream in colour. If pellet is significantly red, repeat washing step again.
Add 5 ml of Buffer B and 500 µl of 10% SDS to pellet. Re-suspend pellet by vortexing vigorously for 30-60 seconds. Then add 50 µl of Proteinase K solution (20mg/ml). The Proteinase K solution should be made fresh and refrigerated prior to use.
Leave to incubate for two hours at 55oC in a water bath. Remove samples and leave to cool to room temperature (or leave for 2-3 minutes on ice). Add 4 ml of 5.3 M NaCl solution. Vortex gently for 15 seconds.
Spin at 4500 rpm for 15-20 minutes at 4oC. Pour off supernatant into a fresh tube. Take care not to dislodge pellet. Add an equal volume of cold isopropanol (stored at -20oC). Invert 5-6 times gently to precipitate DNA.
Remove DNA with a wide bore tip and transfer to a microfuge tube. Wash with 1 ml of 70% ethanol. Leave DNA to dry for 15-20 minutes at 37oC. Re-suspend in 300-400 µl of Tris HCl, pH 8.5 (not TE!). Leave to re-dissolve overnight at room temperature. DNA can be safely refrigerated for up to a year. Long-term storage may involve ethanol at -70oC.
i have some questions about it
1- how much volume of blood can i start with???
2- is it applied to start with 1 ml blood???? if so, can i still use the same quantities of the other reagents in the protocol?????
3- how can i prepare Tris-HCl pH 8.5 (in whicj re-dissolve DNA at final step)?????
Thank u very much. best regards.
1. I think you may wish to start with 1 ml of blood.
3. You may need to find out the concentration of Tris HCl.
Hope this help.
The protocol seems to be designed to give you DNA from blood, the first step is just washing the blood cells so you could start with more than 1 ml. Personally I think the protocol is designed for 5-10 ml of blood, just from the volumes of reagents given, with one ml of blood you won't get enough cells to have enough DNA to efficiently precipitate in the large volumes used in the subsequent steps. Having said that, you will still get some DNA, just not much.
I would recommend 10 mM Tris-HCl pH 8.5, dissolve 1.2114 g of Tris base in 900 ml of water, adjust pH to 8.5 with 1 M (or conc, won't take much though) HCl, and make up to 1.0 L, autoclave.