Sam:LabNotes/Human/2009-5-22

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Revision as of 20:53, 26 May 2009 by >Sam Chiang (→‎Purification)
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2nd MDA amplicons purification using MICROCON kit

Objective

  • In order to estimate the DNA concentration of 2nd MDA amplicons. 20uL of 2nd MDA amplicon were purified using MICROCON kit(#YM-100, Millipore) and then measured dsDNA concentration using Nanodrop.
  • It is suggesed to purify the MDA amplicon before nanodrop measuring. Otherwise the primer dimmer and salts in MDA amplicon might interfere the real dsDNA concentration.
  • Only 20uL 2nd MDA amplicon was purified and measured. Since the purification of whole 2nd MAD amplicon (200uL) is not effective and time consuming.


Samples

  • 200uL of 2nd MDA amplicons from:
    • 2ndMDA-L4N9(8hr), 2ndMDA-L4N9(4hr), 2ndMDA-L4N8(6hr), 2ndMDA-L4N8(4hr), 2ndMDA-L4N7(6hr), 2ndMDA-L4N7(4hr), 2ndMDA-N6(6hr) and 2ndMDA-N6(4hr)


Purification

  • Transfer 20uL of MDA amplcion into a 1.5-mL tube.
  • Add Nuclease-free H2O (Ambion) up to 200uL of total volume. Mix by vortexing for 10 sec and briefly spining down the mixture.
  • Assemble the filter
  • Transfer the mixture on the filter reservoir.
  • Cap the tube and centrifuge at 14,000xg for 12 min.
  • Add 20uL Nuclease-free H2O (Ambion) on the filter. Stay the filter for one min in RT. Invert and transfer the filter on a fresh collection tube. Centrifuge at 3000xg for 3 min.
  • Estimate the volume of elutant using tips. Nanodrop the dsDNA concentration.

NOTE: In order to know the general recovery rate of Mocrocon kit, the purified went through the 2nd Microcon purification and the new DNA concentration and volumn were recorded.

Results

Discussion