E.Z.N.A. ® Blood Miniprep DNA kit Protocol (PDF Version)

Vortex thoroughly to mix.Problem Possible Cause SuggestionsTip: For example, if only 200 l of clear supernatant is obtained, add 200 l Buffer BLfollowed by 200 l isopropanol.®5. Add the mixture to a HiBind DNA mini column assembled in a 2 ml collection tube andproceed with the basic Blood DNA protocol from Step 8 (Page 4).Blood spots from finger pricks usually contain no more than 50 l blood and yield approximately500 ng to 1 g DNA. This is usually sufficient for PCR analysis. To obtain higher DNAconcentrations, elute with 50 l preheated Elution Buffer or TE and repeat with the first eluate.D. Buffy CoatThe buffy coat fraction of whole blood is enriched with W BC, and usually gives at least 5-fold moreDNA than the same volume of blood. To prepare buffy coat from fresh whole blood, simplycentrifuge the sample at 3,000-4,000 x g for 10 min at room temperature. Three layers should beobtained with plasma in the upper layer, leukocytes in the middle layer (buffy coat), anderythrocytes in the bottom layer. Carefully aspirate the plasma, making sure not to disturb thelayer of concentrated leukocytes. The buffy coat can be drawn off with a pipette and used directly® oin the E.Z.N.A. Blood DNA Protocol or frozen at -70 C for storage.Determination of Yield and QualityThe total DNA yield can be determined by a spectrophotometer using deionized water, Tris-HClbuffer, or Elution Buffer as blank. Dilute the DNA in TE buffer and calculate concentration as:[DNA] = (Absorbance260) x (0.05 g/l) x (Dilution factor)The quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm.A ratio of (A /A ) of 1.7-1.9 corresponds to 85%-95% purity.260 280Expected yields range from 4 g to 12 g DNA per 250 l whole blood, depending on source ofsample, its age, and the method of storage. Yields are generally 5-fold higher with Buffy Coatsamples.Troubleshooting GuideProblem Possible Cause SuggestionsPoor elutionImproper washingBuffy Coat usedLow A 260/A 280 ratio Extended centrifugationduring elution step.No DNA ElutedW ashing LeavesColored Residue InColumnPoor cell lysis due toincomplete mixing withBuffer BLHemoglobin remains oncolumnPoor cell lysis due toimproper mixing withBuffer BL.Absolute ethanol notadded to Buffer BL.No ethanol added toW ash BufferConcentrate.Incomplete lysis due toimproper mixing withBuffer BL.Repeat elution or increase elution volume(see note on page 4).oIncubation of column at 70 C for 5 min withElution Buffer may increase yields.W ash Buffer Concentrate must be dilutedwith absolute (100%) ethanol as specifiedon page 5 before use.W ith Buffy Coat samples, use absoluteethanol rather than isopropanol in step 6,page 4.Resin from the column may be present ineluate. Avoid centrifugation at speedshigher than specified. The material can beremoved from the eluate by centrifugation— it will not interfere with PCR or restrictiondigests.Repeat the procedure, this time makingsere to vortex the sample with Buffer BLim m ediately and com pletely.After application of sample to column, washonce with 300 l Buffer BL.Mix thoroughly with Buffer BL prior toloading HiBind column.Before applying sample to column, analiquot of Buffer BL/ethanol must be added.See protocol above.Dilute W ash Buffer with the indicatedvolume of absolute ethanol before use.Buffer BL is viscous and the sample mustbe vortexed thoroughly.Clogged Column Incomplete lysis Add the correct volume of Buffer BL andoincubate for specified time at 70 C. It maybe necessary to extend incubation time by10 min.Sample too largeIf using more than 250 l of blood, increasevolumes of OB Protease/Proteinase K,Buffer BL, and isopropanol. Pass aliquots oflysate through one column successively.Eluted MaterialHas Red/BrownColorNo ethanol added toW ash BufferConcentrate.Sample volume toolarge.Hemoglobin remains oncolumn.Dilute W ash Buffer with the indicatedvolume of absolute ethanol before use.Reduce sample volume and followdirectionsAfter applying sample, wash column oncewith 300 l Buffer BL.Sample too viscousDivide sample into multiple tubes, adjustvolume to 250 l with 10 mM Tris-HCl.Low DNA Yield Clogged colum n See abovePage 7 of 8Page 8 of 8