Revision as of 21:25, 27 March 2015 by >Andrew
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
In tube validation for ALS protocol 3/30/2015
In tube validation for HLS protocol 3/33/2015