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 | ||