Quick Spin High Capacity Columns Troubleshooting

Product No. 03117928001

Troubleshooting

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

Possible reasons would be:

  • Sample should not be applied to the side of the column, so that molecules flow around the matrix instead of through the matrix (without purification).
    Recommendation: Apply sample directly to center of the column bed.
  • 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.
  • The incorrect g-force was used during centrifugation, so that 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.
  • Wrong rotor was used during centrifuge.
    Recommendation: Use swinging-bucket rotor. A fixed-angel rotor may cause nucleotide contamination of final sample.
  • 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 Columns or Quick Spin Columns

Possible reasons for poor sample recovery:

  1. Centrifugation speed is too fast or the centrifugation time is too short: Do not centrifuge the columns faster than recommended speed.
  2. Matrix is not evenly resuspended prior to packaging 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 resuspend 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). Recommendation: 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. Sample amount is too much. 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.


Nonreproducible elution volumes by Quick Spin Columns for radiolabeled DNA purification

Variable elution volumes can produce problems during centrifugation. Only the maximum sample volume of 100 μl should be applied to the G50 columns; an elution volume of 450 μl is only possible when residual matrix suspension buffer is present. Residual buffer from the gel matrix will lead to dilution of the sample and lower the specific activity of the eluate. To resolve eluate variability, address the following:

  1. Is the centrifuge exactly calibrated and balanced? Is it a swing-bucket rotor ?
    When the g-force is too low, then the suspension buffer will not be removed completely. When the centrifuge is not balanced correctly, all the columns will not be centrifuged identically. It is essential to use swinging bucket rotors instead of fixed-angle rotors. Take care that the column is positioned vertically in the centrifuge. A fixed-angle rotor will not achieve the necessary g-force.
  2. The most critical point for a reproducible and high recovery is that the sample is applied to the middle of the gel surface and that the volume of the sample is low enough to ensure that no sample will pass between the matrix and the column wall. It is also important that the column be continuously placed in an upward position during sample application to positioning in the centrifuge.
  3. Do not apply a higher than recommended sample volume (i.e., 50 μl).

Materials

     
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