Andrew:Notebook/AR150618 C1: Difference between revisions

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>Andrew
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==31 nL MDA on C1 with GM12878==
==31 nL MDA on C1 with GM12878==


===Overview===
====Motivation====


====Motivation====
Direct Tn5 on single cells did not yield low-noise libraries; not sure where the bias could be coming from. Best guess at this point is chromatin accessibility. I am therefore going back to a pre-library prep amplification approach using Phi29 in a limited volume.


====Goal====
====Goal====
Test 31 nL MDA followed by Tn5-059 and protease on chip, with off-chip PCR.
Protocol Step/chamber Reagents Scratch C1 Tube
1 ALS g DNA 6.0E-12 1.3E-09
2 NS M Tn5 7.0E-08 7.0E-08
3 MDA mix L rxn 3.2E-08 7.0E-06
4 Tn5 mix mol Tn5 2.2E-15 4.9E-13
5 ProtQ mix # Tn5 1.3E+09 2.9E+11
mol/g 3.7E-04 3.7E-04
fmol/ng 367.50 367.50
Conc adjust. Already filled Load chamber(s) Add rgts at
Capt 1 1.5X
Already filled Load chamber(s) Add rgts at
Capt+1 2 2.5X
Already filled Load chamber(s) Add rgts at
Capt+1+2 3 3.5X
Already filled Load chamber(s) Add rgts at
Capt+1+2+3 4 1.2X
Already filled Load chamber(s) Add rgts at
Capt+1+2+3+4 5 2.2X
Volumes Chamber/Step C1 IFC (nL) Running (nL) In-Tube (uL) Running (uL)
C 4.5 4.5 1 1
E1 9 13.5 2 3
E2 9 22.5 2 5
E3 9 31.5 2 7
E4 135 166.5 30 37
E5 135 301.5 30 67
Total 301.5 67


===Protocol===
===Protocol===

Revision as of 15:52, 9 September 2015

31 nL MDA on C1 with GM12878

Motivation

Direct Tn5 on single cells did not yield low-noise libraries; not sure where the bias could be coming from. Best guess at this point is chromatin accessibility. I am therefore going back to a pre-library prep amplification approach using Phi29 in a limited volume.

Goal

Test 31 nL MDA followed by Tn5-059 and protease on chip, with off-chip PCR.

Protocol Step/chamber Reagents Scratch C1 Tube 1 ALS g DNA 6.0E-12 1.3E-09 2 NS M Tn5 7.0E-08 7.0E-08 3 MDA mix L rxn 3.2E-08 7.0E-06 4 Tn5 mix mol Tn5 2.2E-15 4.9E-13 5 ProtQ mix # Tn5 1.3E+09 2.9E+11 mol/g 3.7E-04 3.7E-04 fmol/ng 367.50 367.50

Conc adjust. Already filled Load chamber(s) Add rgts at Capt 1 1.5X Already filled Load chamber(s) Add rgts at Capt+1 2 2.5X Already filled Load chamber(s) Add rgts at Capt+1+2 3 3.5X Already filled Load chamber(s) Add rgts at Capt+1+2+3 4 1.2X Already filled Load chamber(s) Add rgts at Capt+1+2+3+4 5 2.2X

Volumes Chamber/Step C1 IFC (nL) Running (nL) In-Tube (uL) Running (uL) C 4.5 4.5 1 1 E1 9 13.5 2 3 E2 9 22.5 2 5 E3 9 31.5 2 7 E4 135 166.5 30 37 E5 135 301.5 30 67 Total 301.5 67

Protocol

File:Protocol Worksheet AR150618 C1 v1-1.png File:Protocol Worksheet AR150618 C1 v1-2.png File:Protocol Worksheet AR150618 C1 v1-3-test.png File:ThermalProfile.png

Reagents

File:AR150618 reagents-1.png

Results

qPCR

Gels

File:AR150618 C1 gels.png

Sequencing