Kit Contents:

Notes:

• All procedures should be done at room temperature.
• Prepare a 60°C (and an optional 70°C) water bath.
• Add 180 ml of 98% ethanol to WS Buffer bottle when first Open.
• RNA may also co-purify with genomic DNA, co-purified RNA will not inhibit PCR reaction, but may inhibit some downstream enzymatic reactions. If RNA-free genomic DNA is required, add 50µl of 50 mg/ml RNase A (DNase free) to the sample.
  
  

Protocol:

• Pipette 1 ml sample into a 15 ml tube. Samples: Whole blood, plasma, serum, buffy coat, body fluids, or 10⁷-10⁸ lymphocytes in 1 ml PBS.
  If the sample volume is less than 1 ml, add the appropriate volume of PBS to make up 1 ml.
  
  
• Add 1.2 ml ddH2O to the Proteinase K powder tube (provided) and vortex for 1 minute to completely dissolve Proteinase K.
  The completely dissolved Proteinase K should look transparent, if the tube looks turbid, keep vortex until complete resolution of Proteinase K. The concentration of dissolved Proteinase K is 25 mg/ml.
  
  
• Add 10µl Proteinase K and 1 ml EX Buffer to the sample. Mix immediately by vortexing for 20 seconds.
  If sample volume is larger than 1 ml, increase the amount of EX Buffer and Proteinase K proportionally. Do not add Proteinase K directly to EX Buffer and store dissolved Proteinase K at 4˚ C.
  
  
• Incubate at 60˚C for 20 minutes to lyse the sample, then turn the incubator to 70˚C and incubate 20 minutes. Vortex or invert mix the sample every 3~5 minutes during incubation. Alternatively, place the sample to another 70˚C incubator and incubate for 20 minutes. Sample after complete lysis should not contain insoluble residues and appear viscous.
  
  
• Preheat ddH2O or 10 mM Tris-HCl, pH9.0 to 70C (2.5 ml /prep) for DNA elution.
  
  
• Add 1,050µl of isopropanol or ethanol (96-100%) to the 70˚C-incubated sample of step 4 and mix by vortexing.
  If the sample volume is larger than 1 ml, increase the amount of isopropanol or ethanol proportionally.
  
  
• Place a Genomic DNA Midi Column in a 15 ml Collection Tube (provided). Apply all the mixture from step 6 to the Genomic DNA Midi Column, and centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 3 minutes. Decant the filtrate in the 15 ml tube, and place the Genomic DNA Midi Column back to the tube.
  If a precipitate formed from step 6, apply the precipitate and mixture to the Genomic DNA Midi Column. If Genomic DNA Midi Column clogged after 3 minutes spin, centrifuge again at full speed for another 3 minutes.
  
  
• Add 2.5 ml of WS Buffer. Centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 3 minutes, and discard the filtrate.
  Add 180 ml of ethanol (96-100%) when first open the WS Buffer bottle.
  
  
• Add another 2.5 ml of WS Buffer. Centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 3 minutes, discard the filtrate, and at full speed (about 4,000 rpm) for a further 5 minutes to dry the column.
  
  
• Place the Genomic DNA Midi Column in a new 15 ml tube (provided by user), and discard the Collection tube contains the filtrate.
  
  
• Elute the DNA with 1 ml of preheated ddH2O or 10 mM Tris-HCl, pH 9.0 from step 5. Centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 10 minutes.
  Incubate the 1 ml ddH2O or TE loaded column-tube 5 minutes at 70oC will increase DNA yield.
  
  
• Store eluted DNA at -20˚ C.
  EDTA in TE elution buffer may inhibit PCR reaction, use ddH2O elution for PCR is recommended.
  
  

Troubleshooting:

• Incomplete digestion of Hemoglobin by Proteinase K.
  Prepare a new sample, add 20µl (double amount) of Proteinase K stock (25 mg/ml) to 1 ml EX Buffer and vortex thoroughly, then incubate for 1 hour at 60˚C to completely digest Hemoglobin.
• No alcohol added to the sample before loading onto the Genomic DNA Midi Column.
  Repeat the procedure with a new sample.
• Incorrect amount of ethanol added to the WS Buffer.
  
  

• Too low concentration of sample used.
  Increase the sample volume and repeatedly load into the Genomic DNA Midi Column.
• Incomplete cell lysis due to insufficient mixing of the sample with EX Buffer.
  Thoroughly vortex the sample with EX Buffer.
• No alcohol added to the sample before loading onto the Genomic DNA Midi Column.
  Repeat the procedure with a new sample.
• Elution buffer (ddH2O or 10 mM Tris-HCl, pH 9.0) does not be heated to 70˚C.
  Repeat elution with heated ddH2O and incubate for 5 minutes at 70˚C before spin.
• The pH of Tris buffer is too low.
  The pH of 10 mM Tris-HCl must be 9.0.
• Elute the DNA with less than 1 ml of elution buffer.
  Less than 1 ml of elution buffer will reduce yield.
  
  

• Inefficient cell lysis.
  Thoroughly vortex the mixture of sample.
• Inefficient protein degradation.
  After adding Proteinase K, extend the 60oC incubation time.
• No alcohol added to the sample before loading onto the Genomic DNA Midi Column.
  Repeat the procedure with a new sample.
• Incorrect amount of ethanol added to the WS Buffer.