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]