Kun:LabNotes/Haplotyping/2009-2-12: Difference between revisions

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(New page: ==Comparison of MDA using N6, LN6 and N9 primers== *The objective of this experiment is to compare the amplification bias of several variants of MDA protocols: **Control: N6 primer: 30C **...)
 
Line 29: Line 29:
   RepliPhi Phi-29 (100U/ul)      1.0ul        1.0ul
   RepliPhi Phi-29 (100U/ul)      1.0ul        1.0ul
*Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
*Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
  [[Image:2009-02-12-MDA-N6.png|300px]] [[Image:2009-02-12-MDA-LN6.png|300px]]
  [[Image:2009-02-12-MDA-LN6-Tre.png|300px]] [[Image:2009-02-12-MDA-N9-Tre.png|300px]]
  [[Image:2009-02-12-MDA-LN6-37C.png|300px]]

Revision as of 20:51, 13 February 2009

Comparison of MDA using N6, LN6 and N9 primers

  • The objective of this experiment is to compare the amplification bias of several variants of MDA protocols:
    • Control: N6 primer: 30C
    • LN6 (LNA primer): 30C, 37C, 30C + trehalose
    • N9: 30C + trehalose
  • The template DNA is GM20431, 30pg/reaction (10 copies of diploid genome)
  • Two replicates of GM20431 and NTC (20 reactions in total)

Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)

  • ALS: (400mM KOH, 100mM DTT, 10mM EDTA)
    • Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
    • Mix 8ul 5M KOH, 10ul 1M DTT, 102ul nuclease free H2O.
  • NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5):

Set up MDA reactions

  • Perform serial dilution of GM20431 gDNA to 30pg/ul.
  • Perform UV treatment of ALS and NS buffer 10ul/tube for 5 min;
  • Prepare 2X SYBR Green I solution from the 50X stock solution;
  • Mix 1ul template with 1.5ul ALS buffer, RT for 3min;
  • Transfer to cool block;
  • Add 1.5ul NS buffer;
  • Add 16ul Master mix:
                                  x13           x9
                                 Tre(-)        Tre(+)
  Trehalose(1.2M)                  0ul         5.0ul
  10x RepliPhi phi-29 buffer     2.0ul         2.0ul
  1mM primer                     1.0ul         1.0ul
  25mM dNTP                      0.8ul         0.8ul
  2X SYBR Green I                1.0ul         1.0ul
  H2O                            9.7ul         4.7ul
  RepliPhi Phi-29 (100U/ul)      1.0ul         1.0ul
  • Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
  File:2009-02-12-MDA-N6.png File:2009-02-12-MDA-LN6.png
  File:2009-02-12-MDA-LN6-Tre.png File:2009-02-12-MDA-N9-Tre.png
  File:2009-02-12-MDA-LN6-37C.png