Virus DNA/RNA Isolation Kit

Catalog Number NA2019-##

ATTOGENE CORPATION Solutions for Life Science

Table of Contents Introduction ................................................................. 1

Kit Contents ................................................................. 1

Storage and Stability ................................................. 1

Equipment and Reagent Preparation .................... 1

Sample Preparation Test .......................................... 2

Auto Steps for Allsheng Auto-Pure32A .............. 3-4

Auto Steps for Allsheng Auto-Pure96 ................ 5-6

Auto Steps for KingFisher Duo/Duo Prime ...... 6-7

Auto Steps for KingFisher Flex ............................ 7-9

Manual steps for virus DNA/RNA Isolation .... 9-10

Introduction Virus DNA/RNA Kit is designed for rapid purification of viral RNA and DNA from biofluid samples such as serum, plasma and swab samples. This kit has been adapted for use on automatic magnetic particle isolation devices and optimized to be used for isolation of Viral RNA for COVID testing. It can be integrated with the COVID qRT-PCR workflow at testing centers. It is also capable of being used with manual magnetic stands as needed. Kit Contents:

Component Name 50 Preps 200 Preps

Buffer LY 30mL 120mL

Buffer WS 15mL 60mL

Virus Beads 1mL 1mL x 4

Carrier RNA 250 μl 250 μl x 4

RNase-Free H2O (Dissolve Carrier RNA Powder)

300 μl 300 μl x 4

RNase-Free H2O 10mL 140mL

#Number of reactions demonstrated is based on 200μl sample volume. Buffer LY and Buffer WS contains chaotropic salts which are irritants. Please handle with appropriate laboratory safety measures and wear gloves. Storage and Stability: Store Virus Beads at 4–8℃ upon arrival. Freeze and violent centrifugation should be avoided. Carrier RNA is provided with lyophilized powder or liquid. The lyophilized Carrier RNA powder should be dissolved with RNase-Free H2O prior to use; and stored at -20℃. The rest of the kit can be stored at room temperature (15–25℃).

Equipment and Reagent Preparation: 1. The instrument can be sterilized by UV light before use. 2. 75% Ethanol: Need to be prepared by the user. 3. Buffer WS: Ethanol must be added prior to use into the bottle labeled Buffer WS. The

lyophilized Carrier RNA powder should be dissolved with RNase-Free H2O prior to use.

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4. Buffer LY and Buffer WS must be kept away from light. Check Buffer LY for precipitate before use and re-dissolve at 37℃ if necessary.

Sample Preparation: Whole blood

• Centrifuge at 1500×g for 10 min. Incubate on ice for 3-5min, carefully transfer 200μl supernatant to the plate for automatic isolation.

Serum

• Add 200μl serum to the plate for automatic isolation.

Tissue 1. Place up to 20mg tissue in a 2ml microcentrifuge tube each containing stainless steel

bead (refer to the protocol of manufacturer for suitable size).

2. Add 400μl PBS to each tube.

3. Homogenizing the tissue according to instructions of manufacturer.

4. Centrifuge at 12000rpm for 5 min. Carefully transfer 200μl supernatant to the plate for

automatic isolation.

Swab

• For swabs with preservation solution, transfer 200μl supernatant to the plate for automatic isolation.

• For swabs without preservation solution, add 250-300μl PBS or 0.9% NaCl solution to the sample, vortex and incubate for 10min, transfer 200μl supernatant to the plate for automatic isolation.

Broncheoalveolar lavage and Sputum (It is recommended to inactivate virus before DNA/RNA isolation)

• Add 200μl sample to the plate for automatic isolation.

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Automation Steps for Allsheng Auto-Pure32A 1. Take a 96-well plate, add samples and reagents to the 96-well plate according to the

table below. Note: The total volume of each well should not exceed 900μl or it may overflow.

Table 1. Setting of 96-well plate and reagent dosage

Well Samples

/Reagents Volume

(μl) Instructions For

Pre-preparation Kit Notes

Column 1/7

Samples ≤300 Users are required to

add into ___

Buffer LY 600 The reagent has been

pre-packaged

Check Buffer LY for precipitate before use and re-dissolve at 37℃ if

necessary.

Carrier RNA 5 Users are required to

add into

Carrier RNA is provided with lyophilized powder or liquid. The lyophilized Carrier RNA powder

should be dissolved with RNase-Free H2O prior to use and stored at -

20℃.

Column 2/8 Buffer WS 600 The reagent has been

pre-packaged Ethanol must be added prior to use.

Column 3/9

75% Ethanol 600 The reagent has been

pre-packaged ___

Virus Beads 20

If not been pre-packaged, users

should add into by themselves

Virus Beads tend to settle to the bottom, it is very important to

resuspend the beads thoroughly before use.

Column 4/10 75% Ethanol 600 The reagent has been

pre-packaged ___

Column 5/11 ___ ___ ___ ___

Column 6/12 RNase-Free

H2O 60

The reagent has been pre-packaged

The volume of elution buffer can be adjusted according to requirements.

Add at least 60μl RNase-Free H2O.

2. Start the instrument and set the procedure according to Table 2.

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3. Place a new clean magnetic rod’s tip in the instrument (Be sure to replace the magnetic rod’s tip with a new one, or it will cause cross-contamination of the sample). Place the 96-well plate containing the sample and reagent into the instrument, corresponding to the magnetic rod. Note: Remember to place the magnetic rod’s tip, or the reagent will erode the magnetic bar, affecting the performance of the instrument and subsequent experiments.

4. Collect DNA/RNA: Remove the 96-well plate and magnetic rod’s tip. Transfer the cleared supernatant of Column 6/12 into a new tube and store at -20℃ or -80℃.

Table 2. Program Setting of Allsheng Auto-pure 32A (Magnetic Setup is set to reciprocae magnetsing)

Well Name

Mix Time (min)

Magnet

(sec)

Wait Time (min)

Volume (ul)

Mix Speed (1-10)

Temp (℃)

Mix pos (1-100%)

Mix amp (1-100%)

Magnet pos

(1-100%)

Magnet speed (1-10)

3 Collect 0.2 45 0 600 8 OFF 0 80 0 1

1 Binding 8 60 0 900 8 OFF 0 80 0 1

2 Wash 1 1 60 0 600 8 OFF 0 80 0 1

3 Wash 2 1 45 0 600 8 OFF 0 80 0 1

4 Wash 3 1 45 2.5 600 8 OFF 0 80 0 1

6 Elution 5 90 0 60 8 OFF 0 80 0 1

4 Drop 0.2 0 0 600 5 OFF 0 80 0 1

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Automation Steps for Allsheng Auto-Pure96:

1. Take 96-well plates, add samples and reagents to 96-well plates according to Table 3.

Note: The total volume of each well should not exceed 900μl or it may overflow. Table 3. Setting of 96-well plate and reagent dosage

Step Plate

Position Samples

/Reagents Volume

(μl) Instructions For

Pre-preparation Kit Notes

Binding 3

Samples ≤300 Users are required to

add into ___

Buffer LY 600 The reagent has been

pre-packaged

Check Buffer LY for precipitate before use and

re-dissolve at 37℃ if necessary.

Carrier RNA 5 Users are required to

add into

Carrier RNA is provided with lyophilized powder or liquid. The lyophilized

Carrier RNA powder should be dissolved with RNase-Free H2O prior to use and

stored at -20℃.

Wash 1 4 Buffer WS 600 The reagent has been

pre-packaged Ethanol must be added

prior to use.

Wash 2 5

75% Ethanol 600 The reagent has been

pre-packaged ___

Virus Beads 20

If not been pre-packaged, users

should add into by themselves

Virus Beads tend to settle to the bottom, it is very

important to resuspend the beads thoroughly before

use.

Wash 3 6 75% Ethanol 600 The reagent has been

pre-packaged ___

Elution 7 RNA-Free H2O 60 The reagent has been

pre-packaged

The volume of elution buffer can be adjusted

according to requirements. Add at least 60μl RNase-Free H2O.

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2. Start the instrument, place the 5 plates and the magnetic rod’s tip on the corresponding position in the instrument.

3. Execute procedures (Table 4). 4. Collect DNA/RNA: Remove the 96-well plates and magnetic rod’s tip. Transfer the

cleared supernatant of Elution plate into new tubes and store at -20℃ or -80℃ .

Table 4. Program Setting of Allsheng Auto-pure96

Step Name Plate

Mix Time (min)

Mix amp (%)

Wait Time (min)

Volume (ul)

Mix Speed (1-10)

Temp (℃)

Segment (1-

5)

Cycle Time (1-10)

Magnet

speed

(1-10)

1st Segm

ent time

2nd Segm

ent time

1 Load 6 - - - - - - - - - - -

2 Collect 5 0.2 80 0 600 8 OFF 2 1 1 5 5

3 Binding 3 8 80 0 900 8 OFF 2 1 1 10 10

4 Wash 1 4 1 80 0 600 8 OFF 2 1 1 10 10

5 Wash 2 5 1 80 0 600 8 OFF 2 1 1 5 5

6 Wash 3 6 1 80 2.5 600 8 OFF 2 1 1 5 5

7 Elution 7 5 80 0 60 5 OFF 1 3 1 30 -

8 Unload 6 - - - - - - - - - - -

Automation Steps for KingFisher Duo/Duo Prime:

1. Take a 96-well plate, add samples and reagents to the 96-well plate according to Table 5.

Note: The total volume of each well should not exceed 900μl or it may overflow.

Table 5. Setting of 96-well plate and reagent dosage

Step Well Samples

/Reagents Volume

(μl) Instructions For Pre-preparation Kit

Binding Column A Samples ≤300 Users are required to add into

Buffer LY 600 The reagent has been pre-packaged

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Carrier RNA 5 Users are required to add into

Wash 1 Column B Buffer WS 600 The reagent has been pre-packaged

Wash 2 Column C

75% Ethanol 600 The reagent has been pre-packaged

Virus Beads 20 If not been pre-packaged, users should add into

by themselves

Wash 3 Column D 75% Ethanol 600 The reagent has been pre-packaged

Elution Column E RNA-Free H2O 60 The reagent has been pre-packaged

Leave Tip Column H 12 DP Tip comb - -

2. Start the instrument, place the plate and tip comb on the corresponding position in the instrument.

3. Execute procedures (Table 6). 4. Collect DNA/RNA: Remove the 96-well plate and tip comb. Transfer the cleared

supernatant of Column E into new tubes and store at -20℃ or -80℃.

Table 6. Program Setting of KingFisher Duo/Duo Prime

Step Well

Release Beads Mixing/Heating Parameters Collect Beads

Time Speed Time Speed Heating Temp. Time/Cycle Cycle

Pick up Tip Column H --

Collect Column C 10s Bottom Mix 0.2min Fast -- 30s/ Cycle 2Cycles

Binding Column A 10s Bottom Mix 8min Fast -- 30s/ Cycle 2Cycles

Wash 1 Column B 10s Bottom Mix 1min Fast -- 30s/ Cycle 2Cycles

Wash 2 Column C 10s Bottom Mix 1min Fast -- 30s/ Cycle 2Cycles

Wash 3 Column D 10s Bottom Mix 1min Fast -- 30s/ Cycle 2Cycles

Dry Column D Outside well/tube; 2.5 minutes Elution Column E -- -- 5min Medium -- 30s/ Cycle 3Cycles

Leave Tip Column C -- Automation Steps for KingFisher Flex:

1. Take 96-well plates, add samples and reagents to the 96-well plates according to Table 7.

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Note: The total volume of each well should not exceed 900μl or it may overflow. Table 7. Setting of 96-well plates and reagent dosage

Step Plate Samples

/Reagents Volume

(μl) Instructions For Pre-preparation Kit

Binding Plate 1

Samples ≤300 Users are required to add into

Buffer LY 600 The reagent has been pre-packaged

Carrier RNA 5 Users are required to add into

Wash 1 Plate 2 Buffer WS 600 The reagent has been pre-packaged

Wash 2 Plate 3

75% Ethanol 600 The reagent has been pre-packaged

Virus Beads 20 If not been pre-packaged, users should add

into by themselves

Wash 3 Plate 4

75% Ethanol 600 The reagent has been pre-packaged

Tip Comb - Users are required to replace with a new tip

comb, or it will cause cross-contamination of the sample.

Elution Plate 5 RNA-Free H2O 60 The reagent has been pre-packaged

2. Start the instrument, place the plates and tip comb on the corresponding position in

the instrument. 3. Execute procedures (Table 8). 4. Collect DNA/RNA: Remove the 96-well plates and tip comb from the instrument.

Transfer the cleared supernatant of Plate 5 into new tubes and store at -20℃ or -80℃.

Table 8. Program Setting of KingFisher Flex

Step Plate

Release Beads Mixing/Heating Parameters Collect Beads

Time Speed Time Speed Heating Temp. Time/Cycle Cycle

Pick up Tip Plate 4 --

Collect Plate 3 10s Bottom Mix 0.2min Fast -- 30s/ Cycle 2Cycles

Binding Plate 1 10s Bottom Mix 10min Fast -- 30s/ Cycle 2Cycles

Wash 1 Plate 2 10s Bottom Mix 1min Fast -- 30s/ Cycle 2Cycles

Wash 2 Plate 3 10s Bottom Mix 1min Fast -- 30s/ Cycle 2Cycles

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Wash 3 Plate 4 10s Bottom Mix 1min Fast -- 30s/ Cycle 2Cycles

Dry Plate 4 Outside well/tube; 2.5 minutes Elution Plate 5 -- -- 5min Medium -- 30s/ Cycle 3Cycles

Leave Tip Plate 4 -- Manual steps for virus DNA/RNA isolation:

1. Add 200μl sample into a 2ml microcentrifuge tube. 2. Add 600μl Buffer LY and 5μl Carrier RNA.

Note: If the sample volume is less than 200μl, add the appropriate volume of 0.9% NaCl solution to bring the volume of sample up to a total of 200μl. If the sample volume is more than 200μl, increase the Buffer LY at Buffer LY : Sample=3:1

3. Fully resuspend Virus Beads by vortexing for 1 minute. Add 20μl Virus Beads, and completely resuspend the magnetic particles by vortexing or pipetting up and down for 10 times. Incubate at room temperature for 10 minutes. Mix the tube every 2–3 minutes during incubation to help lysis and binding.

Note: Virus Beads tend to settle to the bottom, it is very important to resuspend the beads thoroughly before use to ensure a homogeneous mix of this reagent is transferred into each tube to avoid the difference between tubes.

4. Place the tube onto a magnetic stand for 1 minute and aspirate the supernatant with pipette carefully without aspirating the magnetic beads.

5. Remove the tube from the magnetic stand and add 600 μl Buffer WS into the tube. Completely resuspend the magnetic beads by vortexing.

Note: Ethanol must be added prior to use into the bottle labeled Buffer WS.

6. Place the tube onto a magnetic stand for 1 minute or until the solution clears. Aspirate the cleared supernatant with a pipette carefully without aspirating the magnetic beads and then discard the supernatant.

7. Remove the tube from the magnetic stand and add 600 μl 75% Ethanol to the tube. Completely resuspend the magnetic beads by vortexing.

8. Place the tube onto a magnetic stand for 1 minute or until the solution clears. Aspirate the cleared supernatant with a pipette carefully without aspirating the magnetic beads and then discard the supernatant.

9. Wash the magnetic beads again by repeating step 7 and 8. 10. Remove any trace of liquid with pipette tips. Air-dry the magnetic beads by placing the

tube at room temperature for 10-20 minutes

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Note: Do not vacuum dry, and excessive drying can lower the recovery rate.

11. Add at least 20μl RNase-Free H2O and resuspend the magnetic beads by pipetting up and down for 10 times or vortexing. Incubate at room temperature for 5 minutes.

12. Place the tube onto a magnetic stand to magnetize the beads. The solution should be cleared after all magnetic beads are completely pelleted.

13. Transfer the cleared supernatant into a new tube.

If none of the above can solve your problem, please contact our technical support department.

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Who we are Attogene is a biotechnology company located in Austin, Texas. Our focus is to enhance health and wellness of plants, animals and the environment by offering and developing customer focused Life Science Products domestically and internationally.

Our mission is to:

• Enhance detection technologies • Enable rapid responses • Enable impactful research discoveries

Contact Us 3913 Todd Lane, Suite 310 Austin, TX 78744

Phone: 512- 333-1330 Email: sales@attogene.com Web: www.attogene.com

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