Revision as of 22:06, 6 May 2015 by >Andrew
C1_OpenApp_150421
Notes
- This protocol uses HLS/Protease followed by Tn5-059
- 25 X Tn5-059 is 17.5 uM --> 1 X = 700 nM
- Illumina has been using 40 nM = .057 X Tn5-059 on 5 ng DNA --> 7e-5 X for 6 pg
- I will use 0.1 X Tn5-059 based on previous experiments
- PCR with Illumina NPM from Nextera kit
- PCR mix loaded into chamber 4, with barcodes backloaded into chamber 5
- Sample: AD 18-99 Full PI NeuN+ cortex sorted 4/8/2015 frozen at 350/uL in 5 uL aliquots
Setup
Reagents
Capture
|
Patient
|
1 rxn (uL)
|
Cell mix |
Nuclei |
5
|
|
C1 suspension reagent |
3.333333333
|
|
Chamber 1
|
Reagent
|
[Initial]
|
[Final]
|
Adjust
|
1 rxn (uL)
|
HLS mix |
H2O |
NA |
NA |
NA |
4.35
|
Volume (uL) |
Tris-HCl (M) |
0.4 |
0.03 |
1.5 |
0.75
|
10 |
Qiagen Protease (mg/mL) |
10 |
4.40 |
1.5 |
4.40
|
|
C1 Loading Reagent (X) |
20 |
1.00 |
1 |
0.50
|
|
|
|
|
Sum (uL) |
10.00
|
|
Chamber 2
|
Reagent
|
[Initial]
|
[Final]
|
Adjust
|
1 rxn (uL)
|
Tn5 mix |
H2O |
NA |
NA |
NA |
2.0
|
Volume (uL) |
Tn5 buffer (X) |
5 |
1.00 |
2.5 |
5.00
|
10 |
Tn5-059 (X) |
1 |
0.10 |
2.5 |
2.50
|
|
C1 Loading Reagent (X) |
20 |
1.00 |
1 |
0.50
|
|
|
|
|
Sum (uL) |
10.00
|
|
Chamber 3
|
Reagent
|
[Initial]
|
[Final]
|
Adjust
|
1 rxn (uL)
|
ProtQ mix |
H2O |
NA |
NA |
NA |
8.05
|
Volume (uL) |
Tris-HCl (M) |
0.40 |
0.03 |
1 |
0.75
|
10 |
Qiagen Protease (mg/mL) |
10 |
0.20 |
3.5 |
0.70
|
|
C1 Loading Reagent (X) |
20 |
1.00 |
1 |
0.50
|
|
|
|
|
Sum (uL) |
10.00
|
|
Chamber 4
|
Reagent
|
[Initial]
|
[Final]
|
Adjust
|
1 rxn (uL)
|
PCR mix |
H2O |
NA |
NA |
NA |
8.38
|
Volume (uL) |
NPM (X) |
3.33 |
1.00 |
2.233 |
20.12
|
30 |
C1 Loading Reagent (X) |
20 |
1.00 |
1 |
1.50
|
|
|
|
|
Sum (uL) |
30.00
|
|
Chamber 5
|
Reagent
|
[Initial]
|
[Final]
|
Adjust
|
1 rxn
|
Primer mix |
H2O |
NA |
NA |
NA |
0.568
|
Volume (uL) |
N7xx (uM) |
1 |
0.2 |
2.233 |
4.466
|
10 |
S5xx (uM) |
1 |
0.2 |
2.233 |
4.466
|
|
C1 Loading Reagent (X) |
20 |
1.00 |
1 |
0.5
|
|
|
|
|
Sum (uL) |
10
|
|
Protocol
[File:Protocol Worksheet HLS_150421 v1.pdf|600px]
Gel
File:C1 OpenApp 150421.jpg