Spinraza on biozen Oligo : Serum load Optimization on micro-Clarity-OTX

LC Conditions

Column

bioZen 2.6 µm Oligo, LC Column 50 x 2.1 mm, Ea

Brand

Biozen LC

Part No

00B-4790-AN

Phase Name

Oligo

Elution Type

Gradient

Mobile Phase

A: Water + 15 mM N,N-diisopropylethylamine + 35 mM hexafluoroisopropanol

B: 90/10 methanol /water + 15 mM N,N-diisopropylethylamine + 35 mM hexafluoroisopropanol

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

80

20

2

5

60

40

3

5.5

10

90

4

7

10

90

5

7.5

80

20

6

10.5

80

20

Flow Rate

0.25 mL/min

Temperature

70°C

System

Agilent Technologies SCIEX 7500 QTRAP

Detection

LC/MS/MS @ (400°C)

Detection Info

Sample Notes

Clarity OTX 2mg micro elution plate

Sample Preparation Details

Description

Clarity OTX 2 mg Plate, 2 mg/well Microelution 96-well plate, Ea

Part No

8M-S103-4GA

Pretreatment Note

1.-Condition: 2x 200µl of methanol 2.-Equilibrate: 2x 200 µl of equilibration buffer 3.-Prep sample/Bind: in a 300 µl glass vial Lysis buffer + Oligo/Serum in a 1:1 Ratio. Up to 10 µL of Serum . Vortex and transfer to well. 4.-Wash/Bind: Add 100 µl of equilibration buffer to glass vial where sample was previously stored, transfer to well, use 2.5Hg to wash/bind. 5.-Wash1: Equilibration buffer 100µl x 2 6.-Wash2: Wash buffer 100µl x 2 7.-Elute: 30 µl of elution buffer x2 for a total of 60µl, elute in a glass coated receiver plate. Inject as soon as sample is eluted

Condition 1

2x 200µl of methanol

Sample Pretreatment

Prep sample right before adding to the well plate to avoid oligo loss to tube, if possible.

Load

rep sample/Bind: in a 300 µl glass vial Lysis buffer + Oligo/Serum in a 1:1 Ratio. Up to 10 µL of Serum . Vortex and transfer to well.

Wash 1

Wash/Bind: Add 100 µl of equilibration buffer to glass vial where sample was previously stored, transfer to well, use 2.5Hg to wash/bind.

Wash 2

Equilibration buffer 100µl x 2

Wash 3

Wash buffer 100µl x 2

Elution 1

30 µl of elution buffer x2 for a total of 60µl, elute in a glass coated receiver plate. Inject as soon as sample is eluted

Evaporation

Evaporate to dryness

Steps

0

Condition

2x 200µl of methanol

1

Equilibrate

Equilibrate: 2x 200 µl of equilibration buffer

2

Load

rep sample/Bind: in a 300 µl glass vial Lysis buffer + Oligo/Serum in a 1:1 Ratio. Up to 10 µL of Serum . Vortex and transfer to well.

3

Wash

Wash/Bind: Add 100 µl of equilibration buffer to glass vial where sample was previously stored, transfer to well, use 2.5Hg to wash/bind.

4

Wash

Equilibration buffer 100µl x 2

5

Wash

Wash buffer 100µl x 2

6

Elute

30 µl of elution buffer x2 for a total of 60µl, elute in a glass coated receiver plate. Inject as soon as sample is eluted