Kun:LabNotes/Haplotyping/2009-1-29

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Perform 2nd round MDA on the amplicons from 01/28/2009

  • The purpose of this experiment is to generate more DNA for downstream analyses. Only the 16 amplicons from the haploid templates will be re-amplified.
    Set up master mix:
                                         x 16
  10x RepliPhi phi-29 buffer     20ul     320
  1uM N6 primer                  10ul     160
  25mM dNTP                      8ul      128
  2X SYBR Green I                10ul     160
  H2O                           142ul    2272
  RepliPhi Phi-29 (100U/ul)      10ul      30
  Add 200ul MDA mix to each of the tube on cool block, 30C 2h, 85C 3min.
  Make aliquots of amplicons (50ul each) for Infinium Genotyping:
  GM18508A
  GM18507B
  GM18507D
  GM18507G
  GM20431A
  GM20431B
  GM20431C
  GM20431F

  Ethanol precipitation of 150ul amplicons:
  GM18508A
  GM18507B
  GM18507D
  GM18507G
  GM20431A
  GM20431B
  GM20431C
  GM20431G

S1 nuclease debranching

  • Resuspend the precipitated DNA in 100ul ddH2O.
    Set up S1 nuclease digestion
    MDA amplicon:                     20ul
    10X S1 nuclease buffer:            5ul
    3M NaCl:                           5ul
    H2O:                              15ul
    S1 nuclease (Invitrogen, 10U/ul):  5ul
    37c 1h -> 2C -> Purified with Qiaquick columns.
    Check the size of debranched DNA with 1% argarose gel.

The DNA are smears of 10kb or less. The gel image was accidentally erased.

DNA shearing

  • I sheared three samples (Cell-a, Cell-b, gDNA-a) with nebulizers at 35psi for 6 min.
  • The sheared DNA (~600ul) were precipitated with 1V isopropanol.
  • The recovery was pretty poor. Also the sheared DNA fragments are still too big (500bp-1.2kb).
  • I decided to send all the amplicons (5 S1 treated, and 11 untreated) to Covaris for shearing. The target size is 300bp.