Andrew:Notebook/AR150618 C1: Difference between revisions
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==31 nL MDA on C1 with GM12878== | ==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==== | ====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