血液DNA快速提取方法

rapid extraction of high quality DNA from whole blood stored at 4c for long periodMaterials and MethodsStandard chemicalsthis method uses standard chemicals that can be obtained from any major supplier；we used chemicals supplied by sigma co . as follow :Edta (0.5m)，ph 8.0: add 186.1 gr of anhy dras EDTA to 800ml of distilled water。adjust ph to 8.0 with NaOH pellets。make up to 1 liter with distilled water。auto clave at 15p.s.i. for 15 min。1 M Tris-HC1，Ph 7.6: dissolve 121.1 gr of Tris base in 800ml of distilled water . adjust Ph with concentrated HCl . allow mixture to cool toauto clave at 15p.s.i. for 15min。preparation of red blood cell lysis buffer 3360 0.01m tris-HCl ph 7.6，320mm sucrose，5mm MGC 12，1% triton x 100。add 10 ml of 1 m tris，109.54 gr of sucrose，1.01 gr MGC 12，adjust ph to 8.0 And finally add 10ml of triton x-100 to 800ml of distilled wattsauto clave at 15p.s.i. for 10 min。sugars at high temperature can cause carameization(brown ing)，which degrades the sugars 5。preparation of nucleic lysis buffer 3360 0.01m tris-HC1，11.4mm sodium citrate，1mm EDTA，1% sodium dodcyl sulphatetake 10 ml of 1 m tris-HC1 (ph 7.6)、3.75 gr of anhydras EDTA (ph 8.0)、10 gr SDS、2.94 gr of sodium citrate、And adjustmake up to 1 liter with distilled water。auto clave 15 min at 15p.s.i .Tebuffer，ph 8.03360 take 5ml of 1m tris-HCl，ph 7.6，2ml of 0.5m EDTA，ph 8，and make up to 1 liter with distilled water。adjust ph to 8.0 and auto clave 15min at 15 . p . s . IChloroform prechilled to 4C。Ethanol (100%) prechilled to -20C。Procedure of DNA ExtractionBefore starting DNA extraction，liquid blood venogicts should be shake gently by rotating blood mixer(vortex)1.pour 500 l of blood into a 1.5ml eppen dorf tube and add 1000 l of red cell lysis buffer。2.shake micro fuge tube gently(up to homogening)，then spin for 2 minutes at 7000 rpm。3.discard super natant and repeat steps 1-3 two or three more times to remove hemoglobin。it is important to breakdown the pellet by vortex ing and rins Es it well in red blood cell lysis buffer in order to clean the white blood cells from resduars4.placing the tube on tissue paper for few seconds downward . be careful from cross-containation between different samples。5.add 400 l of nucleic lysis buffer to eppen dorf 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 chloro form to eppen dorf tube and mix on a rotating blood mixer at room temperature then spin it for 27.传输400 l of supernatant to a new 1.5ml tube。8.add 800 l of cold(-20c)absolute ethanol and shake it gently then vortex it。DNA should appearas a mucus-like strand in the solution phase。9.spin the micro fuge tube for one minute at 12000 rpm to precipitate，Then discard super natant carefully and let tube completed in room temperatures10.Add 50l of TE to it then vortexkeep eppendorf tube of DNA in 4c or-20c for latuses。we route inely use about one l per PCR reaction without adverse affects . DNA can be quantified a Nd diluted to a working concentration at this point or siswe expect that the yifield of this pro