Andrew:Notebook/C1 InTube 23nL MDA 150324
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In tube validation for 23 nL MDA protocol 3/24/2015[edit]
Overview[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 | ||
In tube validation for 23 nL MDA protocol 3/24/2015[edit]
Notes
- This protocol is based on the current method of Tn5 library prep using MIDAS amplicons
- NS is included in MDA mix, saving a step and allowing on-chip PCR
- PCR mix must be backloaded with barcoded primers
- 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) | 20 | Total (uL) |
ALS | H2O | NA | NA | NA | 1.52 | 30.36 | 2 |
KOH (M) | 5 | 0.40 | 1 | 0.16 | 3.20 | ||
DTT (M) | 0.11 | 0.01 | 1 | 0.18 | 3.64 | ||
EDTA (M) | 0.5 | 0.01 | 1 | 0.04 | 0.80 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.10 | 2.00 | ||
Step 2 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 5 | Total (uL) |
NS + MDA | H2O | NA | NA | NA | 0.17 | 0.85 | 2 |
HCl (M) | 2 | 0.40 | 1 | 0.40 | 2.00 | ||
RepliPhi buffer (X) | 10 | 1.00 | 2.5 | 0.50 | 2.50 | ||
dNTPs (mM) | 25 | 0.40 | 2.5 | 0.08 | 0.40 | ||
N6* (mM) | 1 | 0.05 | 2.5 | 0.25 | 1.25 | ||
RepliPhi (ug/uL) | 0.1 | 0.01 | 2.5 | 0.50 | 2.50 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.10 | 0.50 | ||
Step 3 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 2.5 | Total (uL) |
Tn5 | H2O | NA | NA | NA | 0.15 | 0.37 | 2 |
Tn5 buffer (X) | 5 | 1.00 | 3.5 | 1.40 | 3.50 | ||
Tn5-059 (X) | 2 | 0.1 | 3.5 | 0.35 | 0.88 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.10 | 0.25 | ||
Step 4 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 4 | Total (uL) |
Protease | H2O | NA | NA | NA | 27.91 | 111.62 | 30 |
Tris-HCl (mM) | 4000 | 30 | 1 | 0.23 | 0.90 | ||
Qiagen Protease (mg/mL) | 20 | 0.20 | 1.233 | 0.37 | 1.48 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.50 | 6.00 | ||
Step 5 | Reagent | [Initial] | [Final] | Adjustment | 1 rxn (uL) | 2.5 | Total (uL) |
Fill-in + PCR | H2O | NA | NA | NA | 15.77 | 39.43 | 30 |
Advantage 2 PCR Buf. (X) | 10 | 1.00 | 2.233 | 6.70 | 16.75 | ||
dNTPs (X) | 50 | 1.00 | 2.233 | 1.34 | 3.35 | ||
N7xx (uM) | 10 | 0.2 | 2.233 | 1.34 | 3.35 | ||
S5xx (uM) | 10 | 0.2 | 2.233 | 1.34 | 3.35 | ||
Klenow Exo- (U/uL) | 10 | 0.1 | 2.233 | 0.67 | 1.67 | ||
Advantage 2 Pol. Mix (X) | 50 | 1 | 2.233 | 1.34 | 3.35 | ||
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.50 | 3.75 | ||
Sequencing[edit]
Sample | N7xx | S5xx |
222 nuclei, 1-02 Ctx+ Full | N702 | S502 |
NTC | N702 | S503 |
Related experiments[edit]
In tube validation for 31 nL MDA protocol 3/20/2015
- Includes uploaded excel worksheet