Andrew:Notebook/C1 InTube 23nL MDA 150324: Difference between revisions
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>Andrew (Created page with "==In tube validation for 23 nL MDA protocol 3/24/2015== Motivation *The C1 Open App IFC contains 5 chambers, plus the capture site *This places a constraint on protocol desig...") |
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==In tube validation for 23 nL MDA protocol 3/24/2015== | ==In tube validation for 23 nL MDA protocol 3/24/2015== | ||
===Overview=== | |||
Motivation | Motivation | ||
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===In tube validation for 23 nL MDA protocol 3/24/2015=== | |||
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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Step 1''' | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''[Initial]''' | |||
| align="center" style="background:#f0f0f0;"|'''[Final]''' | |||
| align="center" style="background:#f0f0f0;"|'''Adjustment''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''20''' | |||
| align="center" style="background:#f0f0f0;"|'''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|| | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Step 2''' | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''[Initial]''' | |||
| align="center" style="background:#f0f0f0;"|'''[Final]''' | |||
| align="center" style="background:#f0f0f0;"|'''Adjustment''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| align="center" style="background:#f0f0f0;"|'''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|| | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Step 3''' | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''[Initial]''' | |||
| align="center" style="background:#f0f0f0;"|'''[Final]''' | |||
| align="center" style="background:#f0f0f0;"|'''Adjustment''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''2.5''' | |||
| align="center" style="background:#f0f0f0;"|'''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|| | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Step 4''' | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''[Initial]''' | |||
| align="center" style="background:#f0f0f0;"|'''[Final]''' | |||
| align="center" style="background:#f0f0f0;"|'''Adjustment''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''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|| | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Step 5''' | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''[Initial]''' | |||
| align="center" style="background:#f0f0f0;"|'''[Final]''' | |||
| align="center" style="background:#f0f0f0;"|'''Adjustment''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''2.5''' | |||
| align="center" style="background:#f0f0f0;"|'''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=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''N7xx''' | |||
| align="center" style="background:#f0f0f0;"|'''S5xx''' | |||
|- | |||
| 222 nuclei, 1-02 Ctx+ Full||N702||S502 | |||
|- | |||
| NTC||N702||S503 | |||
|- | |||
| | |||
|} | |||
===Related experiments=== | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Andrew:Notebook/C1_InTube_31nL_MDA_150320 In tube validation for 31 nL MDA protocol 3/20/2015] | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php?title=Andrew:Notebook/C1_InTube_ALS_150330&action=edit&redlink=1 In tube validation for ALS protocol 3/30/2015] | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php?title=Andrew:Notebook/C1_InTube_HLS_150333&action=edit&redlink=1 In tube validation for HLS protocol 3/33/2015] |
Revision as of 21:23, 27 March 2015
In tube validation for 23 nL MDA protocol 3/24/2015
Overview
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
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
Sample | N7xx | S5xx |
222 nuclei, 1-02 Ctx+ Full | N702 | S502 |
NTC | N702 | S503 |
Related experiments
In tube validation for 31 nL MDA protocol 3/20/2015