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

In tube validation for ALS protocol 3/30/2015

In tube validation for HLS protocol 3/33/2015