Andrew:Notebook/C1 OpenApp 150421: Difference between revisions

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===Setup===
===Setup===
====Overview====
[[File:Reagents-page-002.jpg|800px]]


====Reagents====
====Reagents====


{| {{table}}
[[File:Reagents-page-001.jpg|800px]]
| align="center" style="background:#f0f0f0;"|'''Capture'''
 
| align="center" style="background:#f0f0f0;"|'''Patient'''
[[File:Reagents.xlsx|Reagent excel file]]
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)'''
 
|-
====Protocol====
| Cell mix||Nuclei||5
|-
| ||C1 suspension reagent||3.333333333
|-
|
|}


{| {{table}}
[[File:Protocol_Worksheet_HLS_150421_v1-page-001.jpg|800px]]
| 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}}
[[File:Protocol_Worksheet_HLS_150421_v1-page-002.jpg|800px]]
| 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}}
[[File:Protocol_Worksheet_HLS_150421_v1-page-003.jpg|800px]]
| 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}}
===Screening===
| 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}}
====Size Select====
| align="center" style="background:#f0f0f0;"|'''Chamber 5'''
[[File:C1_OpenApp_150421.jpg|800px]]
| 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
|-
|
|}


====Protocol====
===Re-sequencing===


[File:Protocol Worksheet HLS_150421 v1.pdf|600px]
====Size Select====


====Gel====
[[File:C1_OpenApp_150421_gel2.jpg|800px]]
[[File:C1_OpenApp_150421.jpg|400px]]

Latest revision as of 23:57, 6 May 2015

C1_OpenApp_150421[edit]

Notes[edit]

  • 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[edit]

Overview[edit]

File:Reagents-page-002.jpg

Reagents[edit]

File:Reagents-page-001.jpg

Reagent excel file

Protocol[edit]

File:Protocol Worksheet HLS 150421 v1-page-001.jpg

File:Protocol Worksheet HLS 150421 v1-page-002.jpg

File:Protocol Worksheet HLS 150421 v1-page-003.jpg

Screening[edit]

Size Select[edit]

File:C1 OpenApp 150421.jpg

Re-sequencing[edit]

Size Select[edit]

File:C1 OpenApp 150421 gel2.jpg