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==In tube validation for HLS protocol 3/33/2015== 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. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Step/chamber''' | align="center" style="background:#f0f0f0;"|'''31 nL MDA''' | align="center" style="background:#f0f0f0;"|'''23 nL MDA''' | align="center" style="background:#f0f0f0;"|'''Direct Tn5, ALS''' | align="center" style="background:#f0f0f0;"|'''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|| |- | |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chamber(s) already filled''' | align="center" style="background:#f0f0f0;"|'''Going into Chamber(s)''' | align="center" style="background:#f0f0f0;"|'''Added reagents at''' | align="center" style="background:#f0f0f0;"|'''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 |- | |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chamber/Step''' | align="center" style="background:#f0f0f0;"|'''C1 IFC (nL)''' | align="center" style="background:#f0f0f0;"|'''Running (nL)''' | align="center" style="background:#f0f0f0;"|'''In-Tube (uL)''' | align="center" style="background:#f0f0f0;"|'''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|| |- | |}
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