Andrew:Notebook/C1 OpenApp 150421: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Andrew
(Created page with "400px")
 
>Andrew
No edit summary
Line 1: Line 1:
==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====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Capture'''
| align="center" style="background:#f0f0f0;"|'''Patient'''
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)'''
|-
| Cell mix||Nuclei||5
|-
| ||C1 suspension reagent||3.333333333
|-
|
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Chamber 1'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''[Initial]'''
| align="center" style="background:#f0f0f0;"|'''[Final]'''
| align="center" style="background:#f0f0f0;"|'''Adjust'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Chamber 2'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''[Initial]'''
| align="center" style="background:#f0f0f0;"|'''[Final]'''
| align="center" style="background:#f0f0f0;"|'''Adjust'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Chamber 3'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''[Initial]'''
| align="center" style="background:#f0f0f0;"|'''[Final]'''
| align="center" style="background:#f0f0f0;"|'''Adjust'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Chamber 4'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''[Initial]'''
| align="center" style="background:#f0f0f0;"|'''[Final]'''
| align="center" style="background:#f0f0f0;"|'''Adjust'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Chamber 5'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''[Initial]'''
| align="center" style="background:#f0f0f0;"|'''[Final]'''
| align="center" style="background:#f0f0f0;"|'''Adjust'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|
|}
====Gel====
[[File:C1_OpenApp_150421.jpg|400px]]
[[File:C1_OpenApp_150421.jpg|400px]]

Revision as of 22:03, 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

Gel

File:C1 OpenApp 150421.jpg