AlanFung:LabNotes/CTCF/2011-6-20: Difference between revisions
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*Only Sample A (50ng) gets amplified, may be we need longer amplification time? | *Only Sample A (50ng) gets amplified, may be we need longer amplification time? | ||
*Alice suggests to run a quantification MDA | *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== | ==Nextera Library Construction== | ||
==Sanger Sequencing== | ==Sanger Sequencing== |
Latest revision as of 22:43, 22 June 2011
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