Nucleic Acid Extraction Kit HandBook

VIRAL NUCLEIC ACID

EXTRACTION USER GUIDE
(Version 5.1)

IVD

FOR DIAGNOSTICS USE &

RESEARCH USE ONLY

Catalog No.
SAMPLE: 5 preps
GF-RD-025: 25 preps
GF-RD-050: 50 preps
GF-RD-100: 100 preps
GF-RD-300: 300 preps

High Yield and Purity

Fast and Easy purification
Reliable and Reproducible
Eluted nucleic acid ready for use in downstream applications
No toxic or organic-based extraction required
 GF-1 Viral Nucleic Acid
Extraction Kit
For isolation of viral nucleic acid (DNA/RNA)
from biological samples

Version 5.1

MANUFACTURER AUTHORIZED EUROPEAN

Vivantis Technologies Sdn Bhd REPRESENTATIVE
Headquarters Manufacturing Obelis s.a.
Revongen Corporation Center Vivantis Technologies Sdn Bhd Bd Général Wahis 53
Level 17, Top Glove Tower, Level 1, Enterprise 2, 1030 Brussels, Belgium
No. 16, Persiaran Setia Dagang, Technology Park Malaysia,
Setia Alam, Seksyen U13, Lebuhraya Puchong-Sg. Besi, Tel: +(32)2732-59-54
40170 Shah Alam, 57000 Bukit Jalil, Fax: +(32)2732-60-03
Selangor Darul Ehsan, Malaysia. Kuala Lumpur, Malaysia Email: [email protected]
Tel: +6 03 3359 1166
Fax: +6 03 3358 0303
Email: info @ vivantechnologies.com
Website : www.vivantechnologies.com
Introduction

The GF-1 Viral Nucleic Acid Extraction Kit is designed for rapid and efficient purification of
viral DNA/RNA from samples such as serum, plasma, body fluid or virus-infected cell culture
supernatant. The purification is based on the usage of denaturing agents to provide efficient
viral lysis, denaturation of proteins and subsequent release of DNA or RNA. Special buffers
provided in the kit are optimized to enhance the binding of DNA or RNA onto a
specially-treated glass filter membrane for efficient recovery of highly pure DNA or RNA.

Intended Use

The GF-1 Viral Nucleic Acid Extraction Kit is designed for isolation and purification of viral
DNA/RNA from biological samples such as serum, plasma, body fluid, saliva, biological swabs
or biological cells in VTM and virus-infected cell culture supernatant. For research and in vitro
diagnostic purpose only.

Principle of Test

The GF-1 Viral Nucleic Acid Extraction Kit consists of different special buffers and GF-1
columns with silica-based membrane. The simple 4-step procedure that involves lysis, binding,
washing and elution is designed for efficient isolation of nucleic acid (DNA/RNA) from a broad
range of viruses. Nevertheless, different virus species will have different yield performance.
Procedures must be optimized by the user.

Quality Control

Each lot of GF-1 Viral Nucleic Acid Extraction Kit has been tested against predetermined
specifications to ensure consistent product quality under ISO13485:2016 and ISO9001:2015 –
certified Quality Management System.
Kit components

Product 5 Preps 25 Preps 50 Preps 100 Preps

Catalog No. SAMPLE GF-RD-025 GF-RD-050 GF-RD-100

Components
GF-1 columns 5 25 50 100
Collection tubes 5 25 50 100
Buffer VL 1.5ml 6ml 12ml 24ml
Wash Buffer 1 (concentrate)* 1.5ml 7ml 14ml 28ml
Wash Buffer 2 (concentrate)* 1.7ml 9ml 17ml 36ml
Carrier RNA* 0.3mg 0.5mg 1mg 2 x 1mg
Elution Buffer 1.5ml 2 x 1.5ml 8ml 20ml
Proteinase K* 0.26ml 1.3ml 2 x 1.3ml 3 x 1.7ml
Handbook 1 1 1 1

* Please refer to Reconstitution of Solutions and Storage and Stability.

The GF-1 Viral Nucleic Acid Extraction Kit is available as 25, 50 and 100 purifications per kit.

The reagents and materials provided with the kit are for diagnostics use and research
purposes only.

Sample Material
Tested sample types:
1. Blood, serum, plasma (fresh or frozen biological specimen)
2. Body fluid, saliva
3. Biological swabs or biological cells in VTM
4. Virus-infected cell culture supernatant
Storage and Stability
1. Store all solution at 20°C-30°C.
Store Wash Buffer at room temperature with bottle capped tight after use.

2. Proteinase K and Carrier RNA are stable for up to 1 year after delivery when stored at
room temperature or 4°C.
To prolong the lifetime of Proteinase K and carrier RNA, storage at -20°C is recommended.
Carrier RNA solution (after being reconstituted) can only be thawed not more than once.

3. Kit components are guaranteed to be stable for 18 months from the date of manufacture.

4. Buffer VL and Wash Buffer 1 may exhibit salt precipitation due to cold temperature. If this
occurs, simply warm the bottle at 55°C - 65°C with occasional mixing until completely
dissolved.

5. Any remaining Buffer VL which contains Carrier RNA can only be stored at 4°C for not
more than one week.

Chemical Hazard
Buffer VL and Wash Buffer 1 contain guanidine salts which can be harmful when in contact
with skin or swallowed. Always wear gloves and practice standard safety precautions. Do NOT
disinfect guanidine or extraction waste in solutions containing bleach or any form of acid. To
clean any items contaminated with the reagent, simply soak in detergent and water to remove
all traces of guanidine before cleaning with bleach or acidic solutions.

Limitation
The GF-1 Viral Nucleic Acid Extraction Kit is intended for in vitro diagnostic purpose and
research use only. The kit is not for the detection, prevention or treatment of a disease. Advise
to do spectrophotometric and gel analysis on the extracted nucleic acid for downstream
application.
Reconstitution of Solutions
The bottle labeled Wash Buffer 1 and Wash Buffer 2 contain concentrated buffer which
must be diluted with absolute ethanol (>95%) before use.

For SAMPLE (5 preps),

Add 1.5ml of absolute ethanol into the bottle labeled Wash Buffer 1.
Add 4ml of absolute ethanol into the bottle labeled Wash Buffer 2.
Add 0.3ml of Elution Buffer into the vial of Carrier RNA and mix well, prepare inaliquots to
avoid repeated freeze-thaw cycles. Store at -20°C.

For GF-RD-25 (25 preps),

Add 7ml of absolute ethanol into the bottle labeled Wash Buffer 1.
Add 21ml of absolute ethanol into the bottle labeled Wash Buffer 2.
Add 0.5ml of Elution Buffer into one of the vials of Carrier RNA and mix well, prepare in
15µl aliquots to avoid repeated freeze-thaw cycles. Store at -20°C.

For GF-RD-50 (50 preps),

Add 14ml of absolute ethanol into the bottle labeled Wash Buffer 1.
Add 40ml of absolute ethanol into the bottle labeled Wash Buffer 2.
Add 1ml of Elution Buffer into one of the vials of Carrier RNA and mix well, prepare in 15µl
aliquots to avoid repeated freeze-thaw cycles. Store at -20°C.

For GF-RD-100 (100 preps),

Add 28ml of absolute ethanol into the bottle labeled Wash Buffer 1.
Add 84ml of absolute ethanol into the bottle labeled Wash Buffer 2.
Add 1ml of Elution Buffer into one of the vials of Carrier RNA and mix well, prepare in 15µl
aliquots to avoid repeated freeze-thaw cycles. Store at -20°C. Store the other vial of Carrier
RNA at -20°C and dissolve in Elution Buffer only prior to use.
Procedures
Reminder
· All steps are to be carried out at room temperature unless stated otherwise.
· Wash Buffer 1 and Wash Buffer 2 (concentrate) have to be diluted with absolute ethanol
before use. Please refer to Reconstitution of Solutions.
· If precipitation forms in Buffer VL, incubate at 55°C - 65°C with occasional mixing until
completely dissolved.

· Pre-set waterbath to 65°C.

· Prepare Buffer VL with Carrier RNA by adding 15μl of Carrier RNA into 200μl of Buffer VL
per sample.

1. Sample lysis
Add 50μl of Proteinase K into 200μl of sample and mix thoroughly. Add 215μl of Buffer VL
(containing Carrier RNA) and mix homogeneously by pulsed-vortexing. Incubate at 65°C for
10 min.

2. Addition of ethanol
Add 280μl of absolute ethanol. Mix immediately and thoroughly.
Mix immediately to prevent any uneven precipitation of nucleic acid due to high local ethanol concentrations.

3. Loading to column
Transfer the sample into a column assembled in the collection tube (provided). Centrifuge at
5,000 x g for 1 min. Discard flow through.

4. Column washing 1
Wash the column with 500μl Wash Buffer 1 and centrifuge at 5,000 x g for 1 min. Discard flow
through.
Ensure that ethanol has been added into the Wash Buffer 1 before use (refer to Reconstitution of Solutions).
5. Column washing 2
Wash the column with 500μl Wash Buffer 2 and centrifuge at 5,000 x g for 1 min. Discard
flow through. Wash column again with 500μl Wash Buffer 2 and centrifuge at maximum
speed for 3 min.
Ensure that ethanol has been added into the Wash Buffer 2 before use (refer to Reconstitution of Solutions).
Perform centrifugation for 3 min to remove ethanol.completely.

6. DNA Elution
Place the column into a clean microcentrifuge tube. Add 30-50μl of Elution Buffer or
nuclease- free water directly onto column membrane and stand for 2 min. Centrifuge at
5,000 x g for 1 min to elute DNA/RNA.

Ensure that the Elution Buffer is dispensed directly onto the center of the membrane for complete elution.
Store DNA at 4°C to -20°C or RNA at -20°C to -80°C.
Troubleshooting
Please note that by not adhering to the recommended protocols, unsatisfactory results related
to yield and quality of DNA/RNA may occur. If problems arise, please refer to the following:

Problem Possibility Suggestions

Low DNA/RNA yield Samples not fresh or not Sample can only be thawed not
properly stored more than once.
Carrier RNA is not added to Prepare Buffer VL with Carrier
Buffer VL RNA as described in the
procedures page.

Low quality of Carrier RNA Ensure that the Carrier RNA is

aliquoted and can only be thawed
not more than once. Please refer to
page 3 for the "Storage and
Stability".

Ensure that any precipitate formed

in Buffer VL is completely
dissolved.

Inefficient nuclease Ensure that Buffer VL is mixed

inhibition during sample lysis homogeneously with the mixture of
step sample and Proteinase K.

Ethanol is not added after Repeat purification with new

sample lysis sample.

Wash Buffer 1 and Wash Please refer to 'Reconstitution of

Buffer 2 are reconstituted Solutions". Repeat purification with
wrongly new sample.
Troubleshooting
Problem Possibility Suggestions

Column is not dried before Ensure that column is spun

addition of Elution Buffer dried maximum speed for
3 minutes after addition of
Wash Buffer 2.

Poor performance RNA degraded Process sample immedia-

of eluted DNA/RNA in tely or sample is stored for
downstream later use, ensure that
applications sample is thawed on ice.

Use disposable plastic-

ware and pipette tips.

Ensure that the purificati-

on is performed in an
RNase-free environment.

Eluted DNA/RNA contains Ensure that the Column

traces of ethanol drying step is carried out
prior to elution.

Low concentration of eluted Reduce the amount of

DNA/RNA Elution Buffer but not less
than 30µl

The amount of added carrier User may optimize the

RNA is inappropriate amount of Carrier RNA
to be added.
 Sample Lysis Addition of ethanol
Add 50µl Proteinase K into 200µ l of
Add 280µ l absolute
sample and mix thoroughly. Add 215µl of
ethanol and mix
Buffer VL (containing Carrier RNA) and
immediately.
mix by pulsed vortexing. Incubate at
65°C,10 min

5,000 x g 5,000 x g
1 min 1 min

Loading to column Centrifuge Column Washing Centrifuge

Transfer sample Discard flow through Add 500µl Wash Buffer 1 Discard flow through
to column

5,000 x g 14,000 x g
1 min 3 min

Column Washing Centrifuge Column Washing Centrifuge

Add 500µl Wash Buffer 2 Discard flow through Add 500µl Wash Buffer 2 Discard flow through

5,000 x g
1 min

Elution Centrifuge
Transfer column to a new Store DNA at 4°C or -20°C
microcentrifuge tube Store RNA at -20°C or -80°C
Add 30-50µl Elution Buffer
or water. Stand for 2 min.

MANUFACTURER AUTHORIZED EUROPEAN

Vivantis Technologies Sdn Bhd REPRESENTATIVE
Headquarters Manufacturing Obelis s.a.
Revongen Corporation Center Vivantis Technologies Sdn Bhd Bd Général Wahis 53
Level 17, Top Glove Tower, Level 1, Enterprise 2, 1030 Brussels, Belgium
No. 16, Persiaran Setia Dagang, Technology Park Malaysia, Tel: +(32)2732-59-54
Setia Alam, Seksyen U13, 40170 Shah Alam, Lebuhraya Puchong-Sg. Besi, Fax: +(32)2732-60-03
Selangor Darul Ehsan, Malaysia. 57000 Bukit Jalil, Email: [email protected]
Tel: +6 03 3359 1166 Kuala Lumpur, Malaysia
Fax: +6 03 3358 0303
Email: info@ vivantechnologies.com
Website : www.vivantechnologies.com

DSGFRD_rev5.1_211220