Andrew:Notebook/C1 OpenApp 150421: Difference between revisions
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====Protocol==== | |||
[File:Protocol Worksheet HLS_150421 v1.pdf|600px] | |||
====Gel==== | ====Gel==== | ||
[[File:C1_OpenApp_150421.jpg|400px]] | [[File:C1_OpenApp_150421.jpg|400px]] |
Revision as of 22:06, 6 May 2015
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]