Sam:LabNotes/Human/2009-5-22

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2nd MDA amplicons purification using MICROCON kit[edit]

Objective[edit]

  • 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[edit]

  • 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[edit]

  • 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 DNA went through the 2nd Microcon purification. The new DNA concentration and volumn information were recorded.


Results[edit]

Sample Name L4N9 (4hr) L4N9 (8hr) L4N8 (4hr) L4N8 (6hr) L4N7 (4hr) L4N7 (6hr) N6 (4hr) N6 (6hr)
Nanodrop - Microcon purification - 1st (ng/uL) 657.00 1074.77 725.41 929.92 727.86 886.12 719.13 778.51
Nanodrop - Microcon purification - 1st (260/280) 1.80 1.84 1.79 1.83 1.81 1.84 1.80 1.86
Nanodrop - Microcon pufirication - 2nd (ng/uL) 517.99 902.60 558.39 683.96 564.82 758.62 550.69 555.16
Nanodrop - Microcon purification - 2nd (260/280) 1.78 1.82 1.76 1.86 1.77 1.84 1.71 1.83


Discussion[edit]

  • By comparing the 1st purification and 2nd purification results, only few DNA were lost during the purification. The recovery rate is high.
  • The value of "2nd MDA- Microcon purified" is much lower than unpurified. Also the MDA production increased with the longer incubation time. (The previous conclusion is opposite based on the DNA concentration from unpurified 2nd MDA amplicons)