Thermo Fisher Scientific MagMAX User Manual

USER BULLETIN

Procedure for viral nucleic acid isolation from 200 µL of saliva sample

Pub. No. MAN0019599 Rev. C.0

Item Source

WARNING! Read the Safety Data Sheets (SDSs)

and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from thermoﬁsher.com/

support.

Product information

This user bulletin describes the procedure to isolate viral nucleic acid from 200 µL of saliva samples (raw or preserved) with the following kits:

• MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Cat. No. A42352, Cat. No. A48310)

• MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit (Cat. No. A48383R)

The procedure for automated extractions performed with the KingFisher™ Flex Puriﬁcation System (KingFisher) are described.

Kits and reagents

MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (up to 200 preparations, when 200 µL of sample is used)

MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (up to 2,000 preparations, when 200 µL of sample is used)

MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit (up to 2,000 preparations, when 200 µL of sample is used)

Fisher BioReagents™ Ethanol, Absolute, Molecular Biology Grade

Nuclease-free Water (not DEPC-Treated) MLS

PBS (1X), pH 7.4 (without calcium and magnesium) 10010023

[1]

, or equivalent

A48383R

BP2818100

BP2818500

BP28184

A42352

A48310

Required materials not supplied

Unless otherwise indicated, all materials are available through

thermoﬁsher.com. "MLS" indicates that the material is available

from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item

Automated nucleic acid extraction system and materials

KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head

KingFisher™ Flex 96 Deep-Well Heating Block 24075430

KingFisher™ 96 Deep-Well Plate

KingFisher™ 96 tip comb for DW magnets

Equipment

Laboratory mixer, vortex or equivalent MLS

Single and multichannel adjustable pipettors (1.00 µL to 1,000.0 µL)

Cold block or ice MLS

Source

5400630

95040450

A48305

A48424

95040455

97002534

A48438

A48414

MLS

Tubes, plates, and other consumables

MicroAmp™ Clear Adhesive Film 4306311

MicroAmp™ Adhesive Film Applicator 4333183

Nonstick, RNase-free microcentrifuge tubes (1.5 mL and 2.0 mL)

Sterile aerosol barrier (ﬁltered) pipette tips thermoﬁsher.

[1]

Available at fisherscientific.com.

thermoﬁsher. com/plastics

com/

pipettetips

Guidelines for saliva collection

• Ensure that there was no eating, drinking, smoking, chewing tobacco, chewing gum, brushing teeth, or use of mouthwash for at least 30 minutes before giving a saliva sample.

• At least 30 minutes before saliva collection, rinse the mouth with water by swishing water for 10 seconds and swallowing the water to rid mouth of debris.

• Use the passive drool technique to pool saliva in the mouth, then drool into a collection device.

• Ensure only saliva is collected by using the passive drool technique, with no coughing or collection of phlegm.

• For saliva collection volume, follow the saliva collection device manufacturers instructions for use.

• For raw saliva, collect at least 1 mL.

For Research Use Only. Not for use in diagnostic procedures.

Prepare raw saliva samples

Before you begin

1. Upon receipt of samples for extractions, dilute the raw saliva

sample 1:1 by adding an equal volume of 1X PBS pH 7.4 (without calcium or magnesium) to the tube and vortex well at maximum speed for 1 minute.

2. Let the diluted raw saliva samples sit and settle for at least 30 minutes at 20°C to 25°C.

Note: Gradually, 2 fractions will form. Do not disturb the layers.

3. (Optional) Centrifuge the diluted raw saliva sample at 1,500 x g (3,000 rpm) for 5 minutes to separate the large debris.

4. Aliquot 200 µL from the top fraction of the diluted raw saliva sample into the Sample Plate.

Note: Pipet slowly to avoid large debris and precipitants from the lower fraction.

Prepare preserved saliva samples

1. Upon receipt of samples for extractions, let the preserved

saliva samples sit and settle for at least 30 minutes at 20°C to 25°C.

Note: In some cases, large debris may start to settle to the bottom. A clear separation may not always be visible.

IMPORTANT! Wash Solution may develop inert white or brown

particulates that ﬂoat in solution. This is not a cause for concern and does not negatively aect performance.

• Determine the number of required reactions based on the number of samples to be processed, plus one Negative Control per plate.

• Prepare fresh 60% Ethanol using Ethanol, Absolute, Molecular Biology Grade and Nuclease-free Water (not DEPC-Treated) for the required number of reactions, sucient for 500 μL per reaction, plus 10% overage.

• Label the short side of each KingFisher™ 96 Deep-Well Plate (4):

Label

Sample plate 1

Wash 1 1

Wash 2 1

Elution plate 1

Note: The tip comb will be placed in the Wash 2/Tip comb plate.

• Mark the Negative Control well on the plate.

Number of plates

2. (Optional) Centrifuge the preserved saliva sample at 1,500 x

g (3,000 rpm) for 5 minutes to separate the large debris.

3. Aliquot 200 µL from the top fraction of the preserved saliva sample into the Sample Plate.

Note: Pipet slowly to avoid large debris and precipitants from the lower fraction.

Extract RNA—Automated method (200‑μL sample input volume)

Automated RNA extraction is performed using the KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head and the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Cat. No. A42352, Cat. No. A48310) or MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit (Cat. No. A48383R) with a sample input volume of 200 µL.

Set up the instrument

(200‑μL sample input volume)

1.1. Ensure that the KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head is set up with the KingFisher™ Flex 96 Deep-Well Heating Block.

IMPORTANT! Failure to use the proper magnetic head and heat block results in lower yields

and potential harm to the instrument.

1.2. Ensure that the MVP_Saliva_200_Flex_V1 program has been downloaded from the instrument product page at www.thermoﬁsher.com and loaded onto the instrument.

2 Procedure for viral nucleic acid isolation from 200 µL of saliva sample User Bulletin

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