Andrew:Notebook/C1 OpenApp 150421: Difference between revisions
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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==== | |||
{| {{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 | ||||