Andrew:Notebook/C1 OpenApp 150421

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Revision as of 22:06, 6 May 2015 by >Andrew (→‎Reagents)
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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