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==


====Motivation====
[[File:AR150618_motivation.pdf|600px]]
 
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 | 600px]]
[[File:Protocol Worksheet AR150618_C1 v1-2.png | 600px]]
[[File:Protocol Worksheet AR150618_C1 v1-3-test.png | 600px]]
[[File:thermalProfile.png | 600px]]
 
====Reagents====
 
[[File:AR150618_reagents-1.png | 600px]]
 
===Results===
 
====qPCR====
 
====Gels====
 
[[File:AR150618_C1_gels.png|600px]]
 
====Sequencing====

Revision as of 15:55, 9 September 2015

31 nL MDA on C1 with GM12878

File:AR150618 motivation.pdf