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mini Quick Spin DNA Columns Troubleshooting

Product No. 11814419001

Troubleshooting

Nucleotide contamination of final sample for mini Quick Spin Columns or Quick Spin Columns

Possible reasons are:

  1. Sample should be applied to the side of the column, so that molecules do not flow around the gel matrix, and instead pass through the matrix (for purification): Recommendation: Apply sample directly to center of the column bed.
  2. Column is overloaded, which causes nucleotides to flow through the matrix. Recommendation: Apply a sample containing 0.02 - 1.0 mg/ml nucleic acid. Apply no more than 50 μl sample to a G-25 column, no more than 100 μl to a G-50 column.
  3. The incorrect g-force was used during centrifugation: Column matrix may collapse and unincorporated nucleotides may pass freely through the column.
    Recommendation: Use 1100 x g for centrifugation spins. Be sure that centrifuge is correctly calibrated.
  4. The incorrect rotor was used in the centrifuge.
    Recommendation: Use swinging-bucket rotor. A fixed-angle rotor may cause nucleotide contamination of final sample.
  5. Column was vortexed too long or too vigorously during matrix resuspension. Recommendation:
  • Do not vortex column longer than 5 seconds.
  • Vortex the column at low speed only.
  • Do not use medium or high speed.

Poor sample recovery of mini Quick Spin Column or Quick Spin Column

Possible reasons for poor sample recovery:

  1. Centrifugation speed too fast or centrifugation time too short: Do not centrifuge the columns faster than recommended speed.
  2. Matrix not evenly resuspended prior to packing step: To fully resuspend the matrix before packing step, do one of the following:
    - Invert column vigorously several times and flick the column sharply to help resuspending the matrix
    - Vortex column gently (5 seconds or less, at low speed)
  3. Tipping of the column, which causes backflow of sample and reduced nucleic acids recovery: Keep the column upright during and after application of sample, especially after centrifugation.
  4. Sample volume too small (<20 μl): Do one of the following:
    - Add 1 x STE buffer to sample until total sample volume is 20 μl
    - After applying sample add 1x STE buffer to the matrix. Total volume applied (sample + STE buffer) must not be greater than the maximum sample volume recommended for the column.
    - At < 0.02 mg/ml DNA/RNA recovery may be low. To improve recovery of diluted samples, add carrier (glycogen, tRNA or sperm DNA) to sample.
  5. There is too much sample. At > 1.0 mg/ml, the sample is viscous and may not migrate through the columns easily, leading to poor recovery and / or contamination with smaller molecules.

Materials

     
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