AlanFung:LabNotes/CTCF/2011-6-20

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MDA Bisulfite Converted Sequencing Library Consturction with N9[edit]

Objective[edit]

  • to create a sequencing library using nextera sample prep kit on MDA amplified bisulfite converted DNA sample.

Protocol[edit]

Bisulfite Conversion of GM12878 50ng 100pg & 600pg DNA with negative control[edit]

' Concentration volume H2O
Tube A 50ng 50ng/ul 1 19
Tube B 100pg 176pg/ul 0.568 19.432
Tube C 600pg 176pg/ul 3.41 16.59
Tube D NTC N/A 0 20
  • Proceed with Zymo column based method with improvement tweak protocol
  • Elute with 11ul EB Buffer

MDA Reaction using N9 for 60mins[edit]

Plate Layout '
1
A 50ng
B 100pg
C 600pg
D NTC
E NTC-DMA
*Add 20ul Enzyme mix to the bisulfite converted DNA
Content Volume per Reaction MM
10X RepliPhi Buffer 3 16.5
100uM N9 Primer 10 55
25mM dNTP 0.8 4.4
RepliPhi Phi29 (100U/ul) 1 5.5
Klenow Exo Minus (10U/ul) 0.5 2.75
H2O 4.7 25.85
    30C 60 min,  4C hold

Page Quantification[edit]

File:ZhangLab 2 2011-06-21 18hr 11min.jpg

  • Only Sample A (50ng) gets amplified, may be we need longer amplification time?
  • Alice suggests to run a quantification MDA
  • Store at 4C overnight
  • Dr. Zhang suggests that we perform inactivation at 95C for 3 mins then store at -20C and wait for the L4N9 set to perform ethanol precipitation together

Nextera Library Construction[edit]

Sanger Sequencing[edit]