Kun:LabNotes/Haplotyping/2009-1-29: Difference between revisions

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(New page: ==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...)
 
 
(6 intermediate revisions by the same user not shown)
Line 10: Line 10:
   RepliPhi Phi-29 (100U/ul)      10ul      30
   RepliPhi Phi-29 (100U/ul)      10ul      30
   Add 200ul MDA mix to each of the tube on cool block, 30C 2h, 85C 3min.
   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.
  GM18507A    Purified DNA (50ul) + 50ul 1X TE
  GM18507B    Purified DNA (50ul) + 50ul 1X TE
  GM18507C    Unpurified DNA (50ul) + 50ul 1X TE
  GM18507D    S1 treated DNA (40ul) + 50ul 1X TE
  GM18507E    Unpurified DNA (50ul) + 50ul 1X TE
  GM18507F    Unpurified DNA (50ul) + 50ul 1X TE
  GM18507G    S1 treated DNA (40ul) + 50ul 1X TE
  GM18507H    Unpurified DNA (50ul) + 50ul 1X TE
  GM20431A    Purified DNA (50ul) + 50ul 1X TE
  GM20431B    S1 treated DNA (40ul) + 50ul 1X TE
  GM20431C    Unpurified DNA (50ul) + 50ul 1X TE
  GM20431D    Unpurified DNA (50ul) + 50ul 1X TE
  GM20431E    Unpurified DNA (50ul) + 50ul 1X TE
  GM20431F    Unpurified DNA (50ul) + 50ul 1X TE
  GM20431G    S1 treated DNA (40ul) + 50ul 1X TE
  GM20431H    Unpurified DNA (50ul) + 50ul 1X TE
==Illumina Infinium genotyping results==
===Alleles on heterozygous SNPs===
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Alleles'''
| align="center" style="background:#f0f0f0;"|'''GM18507A'''
| align="center" style="background:#f0f0f0;"|'''GM18507B'''
| align="center" style="background:#f0f0f0;"|'''GM18507D'''
| align="center" style="background:#f0f0f0;"|'''GM18507G'''
|-
| 0||131607||128075||99430||56922
|-
| 1||22482||11383||13441||2874
|-
| 2||11551||26182||52769||105844
|-
|
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Alleles'''
| align="center" style="background:#f0f0f0;"|'''GM20431A'''
| align="center" style="background:#f0f0f0;"|'''GM20431B'''
| align="center" style="background:#f0f0f0;"|'''GM20431C'''
| align="center" style="background:#f0f0f0;"|'''GM20431F'''
|-
| 0||139685||110445||49761||18786
|-
| 1||22272||22277||3004||815
|-
| 2||20738||49973||129930||163094
|-
|
|}

Latest revision as of 07:22, 18 February 2009

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

  • 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[edit]

  • 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[edit]

  • 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.
  GM18507A     Purified DNA (50ul) + 50ul 1X TE
  GM18507B     Purified DNA (50ul) + 50ul 1X TE
  GM18507C     Unpurified DNA (50ul) + 50ul 1X TE
  GM18507D     S1 treated DNA (40ul) + 50ul 1X TE
  GM18507E     Unpurified DNA (50ul) + 50ul 1X TE
  GM18507F     Unpurified DNA (50ul) + 50ul 1X TE
  GM18507G     S1 treated DNA (40ul) + 50ul 1X TE
  GM18507H     Unpurified DNA (50ul) + 50ul 1X TE
  GM20431A     Purified DNA (50ul) + 50ul 1X TE
  GM20431B     S1 treated DNA (40ul) + 50ul 1X TE
  GM20431C     Unpurified DNA (50ul) + 50ul 1X TE
  GM20431D     Unpurified DNA (50ul) + 50ul 1X TE
  GM20431E     Unpurified DNA (50ul) + 50ul 1X TE
  GM20431F     Unpurified DNA (50ul) + 50ul 1X TE
  GM20431G     S1 treated DNA (40ul) + 50ul 1X TE
  GM20431H     Unpurified DNA (50ul) + 50ul 1X TE

Illumina Infinium genotyping results[edit]

Alleles on heterozygous SNPs[edit]

Alleles GM18507A GM18507B GM18507D GM18507G
0 131607 128075 99430 56922
1 22482 11383 13441 2874
2 11551 26182 52769 105844
Alleles GM20431A GM20431B GM20431C GM20431F
0 139685 110445 49761 18786
1 22272 22277 3004 815
2 20738 49973 129930 163094