Andrew:Notebook/C1 InTube HLS 150333

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In tube validation for HLS protocol 3/33/2015[edit]

Motivation

  • The C1 Open App IFC contains 5 chambers, plus the capture site
  • This places a constraint on protocol design, which must be validated in-tube

Objective

  • Test a number of conditions with a scaled-up volume and select the best condition based on sequencing results

Plan

  • I will start with 1 ul and scale all other volumes proportionately.

The scaling factor is 222 = 1 ul/4.5 nl, which is the actual starting volume of the C1 IFC.

  • I will therefore use ~220 nuclei per condition
  • Because the C1 IFC adds reagents succesively, each step requires a concentration adjustment.
Step/chamber 31 nL MDA 23 nL MDA Direct Tn5, ALS HLS Direct Tn5, HLS
1 ALS ALS ALS HLS + Protease
2 NS NS + MDA NS Tn5
3 MDA Tn5 2nd-st + Tn5 Protease
4 Tn5 Protease Protease Fill-in + PCR
5 Protease Fill-in + PCR Fill-in + PCR
Chamber(s) already filled Going into Chamber(s) Added reagents at C1 Loading Reagent
Capture site 1 1.5X 1X
Capture site 1 + 2 1.25X 1X
Capture site 1 + 2 + 3 1.167X 1X
Capture site 1 + 2 + 3 + 4 1.028X = 1X 1X
Capture site 1 + 2 + 3 + 4 + 5 1.015X = 1X 1X
Chamber(s) already filled Going into Chamber(s) Added reagents at C1 Loading Reagent
Capture site + 1 2 2.5X 1X
Capture site + 1 2 + 3 1.75X 1X
Capture site + 1 2 + 3 + 4 1.088X 1X
Capture site + 1 2 + 3 + 4 + 5 1.047X = 1X 1X
Chamber(s) already filled Going into Chamber(s) Added reagents at C1 Loading Reagent
Capture site + 1 + 2 3 3.5X 1X
Capture site + 1 + 2 3 + 4 1.156X 1X
Capture site + 1 + 2 3 + 4 + 5 1.08X 1X
Chamber(s) already filled Going into Chamber(s) Added reagents at C1 Loading Reagent
Capture site + 1 + 2 + 3 4 1.233X 1X
Capture site + 1 + 2 + 3 4 + 5 1.117X 1X
Chamber(s) already filled Going into Chamber(s) Added reagents at C1 Loading Reagent
Capture site + 1 + 2 + 3 + 4 5 2.233X 1X
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