Andrew:Notebook/C1 InTube 31nL MDA 150320
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In tube validation for 31 nL MDA protocol 3/20/2015[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 | ||
31 nL MDA[edit]
Notes This protocol is based on the current method of Tn5 library prep using MIDAS amplicons Libraries must be harvested before barcoded PCR ALS and NS do not require concentration adjustments Some mixes require volumes that are too small to be pipeted. Use the higher volumes on the far right instead
Step 1 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 10 | Total (uL) |
ALS | H2O | NA | NA | NA | 1.52 | 15.18 | 2 |
KOH (M) | 5 | 0.40 | 1 | 0.16 | 1.60 | ||
DTT (M) | 0.11 | 0.01 | 1 | 0.18 | 1.82 | ||
EDTA (M) | 0.5 | 0.01 | 1 | 0.04 | 0.40 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.10 | 1.00 | ||
Step 2 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 10 | Total (uL) |
NS | H2O | NA | NA | NA | 0.90 | 9.00 | 2 |
Tris-HCl, pH 7.5 (M) | 2 | 0.60 | 1 | 0.60 | 6.00 | ||
HCl (M) | 2 | 0.40 | 1 | 0.40 | 4.00 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.10 | 1.00 | ||
Step 3 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 20 | Total (uL) | ' |
MDA | H2O | NA | NA | NA | 0.04 | 0.76 | 2 | |
RepliPhi buffer (X) | 10 | 1.00 | 3.5 | 0.70 | 14.00 | |||
dNTPs (mM) | 25 | 0.40 | 3.5 | 0.11 | 2.24 | |||
N6* (mM) | 1 | 0.05 | 3.5 | 0.35 | 7.00 | 3 | Add to | |
RepliPhi (ug/uL) | 0.1 | 0.01 | 3.5 | 0.70 | --> | 2.1 | 3.9 | |
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.10 | 2.00 | |||
Step 4 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 2.5 | Total (uL) |
Tn5 | H2O | NA | NA | NA | 19.25 | 48.13 | 30 |
Tn5 buffer (X) | 5 | 1.00 | 1.233 | 7.40 | 18.50 | ||
Tn5-059 (X) | 2 | 0.1 | 1.233 | 1.85 | 4.62 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.50 | 3.75 | ||
Step 5 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 4 | Total (uL) |
Protease | H2O | NA | NA | NA | 27.61 | 110.42 | 30 |
Tris-HCl (M) | 4 | 0.03 | 1 | 0.23 | 0.90 | ||
Qiagen Protease (mg/mL) | 20 | 0.20 | 2.233 | 0.67 | 2.68 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.50 | 6.00 | ||
Off-chip | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 2.5 | Total (uL) |
Fill-in + PCR | H2O | NA | NA | NA | 10.07 | 25.18 | 30 |
Advantage 2 PCR Buf. (X) | 10 | 1.00 | 3.233 | 9.70 | 24.25 | ||
dNTPs (X) | 50 | 1.00 | 3.233 | 1.94 | 4.85 | ||
N7xx (uM) | 10 | 0.2 | 3.233 | 1.94 | 4.85 | ||
S5xx (uM) | 10 | 0.2 | 3.233 | 1.94 | 4.85 | ||
Klenow Exo- (U/uL) | 10 | 0.1 | 3.233 | 0.97 | 2.42 | ||
Advantage 2 Pol. Mix (X) | 50 | 1 | 3.233 | 1.94 | 4.85 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.50 | 3.75 | ||
Sequencing[edit]
Sample | N7xx | S5xx |
222 nuclei, 1-02 Ctx+ Full | N701 | S502 |
NTC | N701 | S503 |
Related experiments[edit]
In tube validation for 23 nL MDA protocol 3/24/2015
In tube validation for ALS protocol 3/30/2015
In tube validation for HLS protocol 3/33/2015