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Quick Spin Columns for radiolabeled RNA purification Troubleshooting

Product No. 11273990001, 11274015001

Troubleshooting

Nucleotide contamination of filtered sample using mini Quick Spin Columns or Quick Spin Columns

Possible reasons are:

  1. Sample should not be applied to the side of the column, so that molecules to flow around the matrix; samples should be placed on top of 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, and no more than 100 μl to a G-50 column.
  3. The incorrect g-force was used during centrifugation: At too high speeds the column matrix can collapse and unincorporated nucleotides may pass freely through the column.
    Recommendation: Use 1,100 x g for centrifugation spins. Make certain that the centrifuge is correctly calibrated.
  4. The inappropriate rotor was used during centrifugation.
    Recommendation: Use swinging-bucket rotor. A fixed-angle rotor may cause nucleotide contamination of the resulting 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 is too fast or the centrifugation time too short: Do not centrifuge the columns faster than the 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. The column was tipped to the side, 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, such as 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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