PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit - Invitrogen

PrepSEQ Sample Preparation Kits 

Protocol 

For use with: 

PrepSEQ Mycoplasma Nucleic Acid Extraction Kit 

PrepSEQ 1-2-3 Nucleic Acid Extraction Kit

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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 

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Part Number 4401253 Rev. C 

02/2010

PrepSEQ Sample Preparation Kits Protocol 

Contents 

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 

Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 

How to use this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 

How to obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 

Protocol PrepSEQ Sample Preparation Kits . . . . . . . . . . . . . . . . . . . . . . 7 

About the kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 

Preparation of sample lysates that contain target DNA . . . . . . . . . . . . . . . . . . . . . . . 9 

Sample preparation considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 

PrepSEQ 1-2-3 protocol for Mycoplasma or MMV detection . . . . . . . . . . . . . . 12 

Prepare small-scale (100 to 2000 µL) lysate (Mycoplasma detection only) . . . . 14 

Prepare large-scale (2 to 10 mL) lysate (Mycoplasma detection only) . . . . . . . . 17 

Extraction of Mycoplasma or Mouse Minute Virus DNA from lysate . . . . . . . . . . . . 20 

Double DNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 

Single DNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 

Appendix A Background and Ordering Information . . . . . . . . . . . . . . . . . . . 31 

Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 

Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 

Appendix B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 

Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 

Chemical alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 

Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 

3

Contents 

4 

PrepSEQ Sample Preparation Kits Protocol

Safety information 


About this Protocol 

Preface 

Note: For general safety information, see this section and Appendix B, “Safety” 

on page 35. When a hazard symbol and hazard type appear by a chemical name or 

instrument hazard, see the “Safety” Appendix for the complete alert on the 

chemical or instrument. 

Safety alert words Four safety alert words appear in Applied Biosystems user documentation at 

points in the document where you need to be aware of relevant hazards. Each alert 

word—IMPORTANT, CAUTION, WARNING, DANGER—implies a 

particular level of observation or action, as defined below: 

IMPORTANT! – Indicates information that is necessary for proper instrument 

operation, accurate chemistry kit use, or safe use of a chemical. 

CAUTION! – Indicates a potentially hazardous situation that, if not 

avoided, may result in minor or moderate injury. It may also be used to alert 

against unsafe practices. 

WARNING! – Indicates a potentially hazardous situation that, if not 

avoided, could result in death or serious injury. 

DANGER! – Indicates an imminently hazardous situation that, if not 

avoided, will result in death or serious injury. This signal word is to be 

limited to the most extreme situations. 

SDSs The Safety Data Sheets (SDSs) for any chemicals supplied by 

Applied Biosystems or Ambion are available to you free 24 hours a day. For 

instructions on obtaining SDSs, see Appendix B on page 37. 

IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems 

or Ambion contact the chemical manufacturer. 

5

Preface 

How to use this guide 

How to use this guide 

Text conventions This guide uses the following conventions: 

6 

Bold text indicates user action. For example: 

Type 0, then press Enter for each of the remaining fields. 

Italic text indicates new or important words and is also used for emphasis. 

For example: 

Before analyzing, always prepare fresh matrix. 

A right arrow symbol () separates successive commands you select from a 

drop-down or shortcut menu. For example: 

Select FileOpenSpot Set. 

Right-click the sample row, then select View Filter View All Runs. 

User attention words Two user attention words appear in Applied Biosystems user documentation. Each 

word implies a particular level of observation or action as described below: 

How to obtain support 

Note: – Provides information that may be of interest or help but is not critical to 

the use of the product. 

IMPORTANT! – Provides information that is necessary for proper instrument 

operation, accurate chemistry kit use, or safe use of a chemical. 

For the latest services and support information for all locations, go to 

www.appliedbiosystems.com. 

At the Applied Biosystems web site, you can: 

Access worldwide telephone and fax numbers to contact Applied Biosystems 

Technical Support and Sales facilities 

Search through frequently asked questions (FAQs) 

Submit a question directly to Technical Support 

Order Applied Biosystems user documents, SDSs, certificates of analysis, 

and other related documents 

Download PDF documents 

Obtain information about customer training 

Download software updates and patches 


About the kits 


Protocol 


Purpose of the kits The PrepSEQ Sample Preparation Kits use magnetic particle-based separation 

technology to extract DNA from Mycoplasma cells or Mouse Minute Virus 

(MMV) isolated from a variety of starting material, such as infected cell cultures 

or Mycoplasma liquid cultures. 

Choosing a preparation 

protocol 

Using this kit, you prepare lysates of samples before you extract the target DNA. 

For samples that contain Mycoplasma, the lysates contain Mycoplasma DNA 

from the free organisms in the culture media and from organisms released from 

the infected host cells. For additional information on kit content, see Appendix A, 

“Background and Ordering Information” on page 31. 

Figure 1 on page 8 shows the process for DNA isolation and detection from 

different types of samples and detection tasks. 

For isolation and purification of Mycoplasma DNA from liquid samples, 

Applied Biosystems recommends that the liquid volume vary from 2 to 10 mL for 

large-scale (final cell number < 10 8 ) extractions and 100 to 2000 µL for smallscale 

(final cell number < 10 7 ) extractions. 

For routine screening and preventive maintenance of cell lines, Applied 

Biosystems recommends using: 

The PrepSEQ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit (PN 

4443789) for samples with a starting volume of 100 µL and cell number of 

≤106 . 

The PrepSEQ Mycoplasma Nucleic Acid Extraction Kit (PN 4409733) for 

samples with a starting volume > 100 µL or a final cell number > 106 . 

For isolation and purification of Mouse Minute Virus DNA from liquid samples, 

Applied Biosystems recommends using the PrepSEQ 1-2-3 Mycoplasma 

Nucleic Acid Extraction Kit. 

7 



About the kits 

8 

Task: Detection of MMV or Mycoplasma 

(Cell lines, cell cultures, or in-process 

samples) 

Prepare samples using 

PrepSEQ 1-2-3 protocol 

(page 11) 

Perform single DNA extraction 

(page 26) 

Sample size: 

2 to 10 mL 

Prepare samples 

using PrepSEQ 

small-scale 

protocol 

(page 13) 

Low cell number 

or 

High viability 

Figure 1 Sample preparation workflows 

Task: Detection of Mycoplasma 

(Production harvest) 

Sample size: 

100 to 2,000 µL 

Prepare samples 

using PrepSEQ 

large-scale 

protocol 

(page 16) 

Cell number and cell viability 

High cell number 

or 

Low viability 

Perform double DNA 

extraction (page 21) 

Perform assay using appropriate Mycoplasma, Myco Scan, or MMV PCR detection kit protocol 




Preparation of sample lysates that contain target DNA 


Sample preparation considerations 

Minimizing cellular DNA and/or RNA in the final extracted DNA is critical to 

Mycoplasma DNA detection. High amounts of cellular DNA and/or RNA cause 

PCR inhibition and high background of the SYBR ® Green I dye signal, reducing 

detection of low copy numbers of targets. Factors that affect levels of cellular 

DNA and/or RNA include: 

Viability of cell culture sample – Use of fresh culture samples increases the 

purity of your extracted target DNA. Avoid conditions such as long-term 

storage at 4 °C (or freezing temperatures). Such temperatures cause 

increased death or lysis of cells, which in turn contributes to additional 

background DNA in samples. 

Cell culture sampling – Avoid taking mucous aggregate material from the 

culture into the sample preparation. This material is very likely chromosomal 

DNA released as a result of cell lysis. 

Cell lysate handling – Treat cells as gently as possible. Be sure to perform 

certain steps at 4 °C where indicated in the procedure. 

9 




Before you begin 

10 

Before starting your sample extraction: 

Prepare the following reagents before their first-time use: 

– Binding Solution – Add 30 mL of 100% isopropanol to the empty 

Binding Solution bottle. Mark the bottle label to indicate that 

isopropanol has been added. 

– Wash Buffer – Add 74 mL of 95% ethanol to the Wash Buffer 

Concentrate bottle, mix well, then mark the bottle label to indicate that 

ethanol has been added. 

– Mixture of 95% ethanol and Lysis Buffer – Prepare 25 mL of a 3:2 

mixture of 95% ethanol and Lysis Buffer (15 mL of 95% ethanol, 10 mL 

of Lysis Buffer) in a 50-mL polypropylene tube. The mixture can be 

stored at room temperature for 6 months. 

Magnetic Particles – Before using, incubate the Magnetic Particles at 37 °C 

for 10 minutes, then vortex the tube at setting #7 until the resuspension is 

complete. 

IMPORTANT! White precipitate occasionally forms in the magnetic particles 

tube. Extraction experiments show that formation of precipitate does not 

affect performance. However, if precipitate forms, incubate the tube at 37 °C 

for 10 minutes, then vortex to completely resuspend the particles. 

Place aliquots of 1✕ PBS on ice. For exact aliquot volumes, refer to the scale 

(large or small) of the extraction. When not in use, store 1✕ PBS at 2 to 8 °C. 

Power on the refrigerated centrifuge to allow it to cool down before use. 

Block heaters: Three incubations are required. Use the procedure shown 

below for the number of heaters available. 

Number of block 

heaters available 

Procedure 

1 1. Set the first heater to 56 °C. 

2. After cellular RNA digestion at 56 °C, reset the heater to 

70 °C. 

3. After cell lysis at 70 °C, reset the heater to 37 °C. 

2 1. Set the first heater to 56 °C. Set the second heater to 70 °C. 

2. After cellular RNA digestion at 56 °C, reset the heater to 

37 °C. 

3 1. Set the first heater to 56 °C. 

2. Set the second heater to 70 °C. 

3. Set the third heater to 37 °C. 


PrepSEQ 1-2-3 

workflow 




The PrepSEQ 1-2-3 workflow for routine screening and preventive maintenance 

of cell lines with a starting volume of 100 µl and a cell number of up to 10 6 is 

shown below. For details, see page 12. 

Step 1: Place 100 µL of sample into a 2-mL tube. 

Step 2: Add 1 µL of 10% SDS, 1 µL of 0.5 M EDTA, and 5 µL of RNase 

Cocktail to 100 µl of the starting sample. For multiple samples, prepare a 

master mix and add 7 µl of the mix to 100 µl of the starting sample. Mix 

well by brief vortexing followed by a brief spin. 

Step 3: Incubate at 56 °C for 15 min. 

Step 4: Add 2 µL of Proteinase K. Mix well by brief vortexing 

followed by a brief spin. 

Step 5: Incubate the tube at 56 °C for 5 min. 

Step 6: Add 240 µL of Lysis Buffer. Vortex for 5 sec, 

then quick-spin for 5 sec. 

Step 7: Incubate at room temperature for 5 min. 

Step 8: Incubate the Magnetic Particles Suspension at 37 °C for 10 min 

followed by vortexing until completely resuspended before use. 

Step 9: Add 30 µL of Magnetic Particles and 200 µL of Binding Solution 

to the sample lysate, and vortex for 5 min at setting #7. 

Quick-spin for 15 sec. 

” Proceed to Step 5 & 6 of the Bind DNA section of the “Single DNA 

extraction workflow” on page 26. 

Figure 1 Sample preparation from 100 µL and cell densities of up to 10 6 using 

the PrepSEQ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit. 

11 




PrepSEQ 1-2-3 protocol for Mycoplasma or MMV detection 

12 

IMPORTANT! Treat cells as gently as possible throughout the procedure. Do not 

freeze the cell culture samples before processing. 

1. Place 100 µL of sample (final cell number: ≤ 10 6 ) into a 2-mL 

microcentrifuge tube. 

2. Add 1 µL of 10% SDS, 1 µL of 0.5 M EDTA, and 5 µL of RNase Cocktail to 

100 µl of the starting sample. For multiple samples, prepare a master mix and 

add 7 µl of the mix to 100 µl of the starting sample. Mix well by brief 

vortexing followed by a brief spin. 

3. Incubate at 56 °C for 15 minutes. 

4. Add 2 µL of Proteinase K. Mix well by brief vortexing followed by a brief 

spin. 

5. Incubate the tube at 56 °C for 5 minutes to digest the cellular RNA. 

6. Add 240 µL of Lysis Buffer. Vortex for 5 seconds, then quick-spin for 5 

seconds. 

7. Incubate the tube at room temperature for 5 minutes. 

8. Incubate the Magnetic Particles Suspension at 37 °C for 10 minutes followed 

by vortexing until completely resuspended before use. 

9. Add 30 µL of Magnetic Particles and 200 µL of Binding Solution to the 

sample lysate, and vortex for 5 minutes at setting #7. Quick-spin for 15 

seconds. 

Next step Proceed to single DNA extraction, step 5 of the Bind DNA section on page 27. 


Small-scale lysate 

preparation workflow 




A small-scale lysate preparation workflow for 100- to 2000-µL samples is shown 

below. For details, see page 14. 

Step 1: Place 100 to 2000 µL of sample into a 2-mL tube. 

Process supernatant: keep on ice 

Step 3: Transfer supernatant to a 2-mL 

tube. Keep on ice. 

Step 6: Centrifuge at 16,000 × g for 15 min. 

Step 7a: Carefully aspirate and discard the 

supernatant. Do not disturb the 

Mycoplasma pellet. 

Step 7b: Transfer the cell fractionation 

lysate from Step 5 to the Mycoplasma 

pellet, then resuspend by pipetting using 

a P1000. Transfer to a new 2-mL tube. 

Step 8: Add 2 µL of 0.5 M EDTA and 

18 µL of RNase Cocktail. Vortex briefly, 

then quick-spin. 


Vortex twice during incubation. 

Step 10: Add 5 µL of Proteinase K. Vortex 

briefly, then quick-spin. Incubate at 56 °C 

for 10 min. 

Step 11: Add 700 µL of Lysis Buffer. Vortex 

for 5 sec at setting #7. 

Total 1 mL of lysate for DNA extraction 

Step 2: Centrifuge at 1000 × g for 3 min. 

Process cell pellet: keep on ice 

Step 4a: Add 1 mL of ice-cold 1✕ PBS. 

Use a P1000 to gently resuspend the cells. 

Step 4b: Centrifuge at 500 × g for 5 min at 

4 °C. Discard the supernatant. 

Step 4c: Add 400 µL of ice-cold Cell 

Fractionation Buffer to the cell pellet. Use a 

P1000 to gently resuspend the cells. Avoid 

cell lysis. 

Step 4d: Incubate on ice for up to 5 min. 

Step 5: Centrifuge at 1000 × g for 3 min at 

4 °C. Transfer 300 µL to a 2-mL tube. Avoid 

the pellet, which contains nuclei and 

mucous aggregate material. Recentrifuge 

as necessary. Keep on ice. 

Figure 2 Preparation of lysate from 100 to 2000 µL of sample using the 

PrepSEQ Mycoplasma Nucleic Acid Extraction Kit; final sample cell 

number ≤10 7 cells. Critical steps are indicated in bold font. 

13 




Prepare small-scale (100 to 2000 µL) lysate (Mycoplasma detection only) 

14 



1. Place 100 to 2000 µL of the cell culture sample (final cell number: ≤ 10 7 ) into 

a 2-mL microcentrifuge tube. 

2. Centrifuge the tube at 1000 × g for 3 minutes to pellet the cells. Place the 

cell pellet on ice to reduce cell lysis. 

3. Transfer the supernatant to a new 2-mL tube. The supernatant contains free 

Mycoplasma organisms in the culture. Keep the supernatant on ice while 

working on the cell pellet; the supernatant is processed at step 6 on page 15.. 

IMPORTANT! If two centrifuges are available, you can centrifuge the 

supernatant at this step while you process the cell pellet in steps 4 through 5. 

4. Process the cell pellet from step 2. 

a. Critical step – Gently resuspend the cell pellet with 1 mL of ice-cold 

1✕ PBS by gentle pipetting with a P1000. 

IMPORTANT! Avoid cell lysis as much as possible. Keep the cells at 

4 °C and treat very gently. Higher temperature increases lysis of the host 

cell nuclei, leading to increased host DNA in the final extracted DNA, 

which causes PCR inhibition. 

b. Critical step – Centrifuge the tube at 500 × g for 5 minutes to pellet the 

cells. Discard the supernatant. 

c. Critical step – Add 400 µL of ice-cold Cell Fractionation Buffer to the 

cell pellet. Very gently pipette up and down several times with a P1000 

to resuspend the cells. 

IMPORTANT! You must perform this step at 4 °C. Room temperature 

increases lysis of the host cell nuclei leading to increased host DNA in 

the final extracted DNA, which causes PCR inhibition. 

d. Critical step – Incubate the cell suspension on ice for 5 minutes. Do not 

exceed 5 minutes. 


increases lysis of the nuclei and host DNA in the final extracted DNA 

and causes PCR inhibition. 





5. Critical step – Centrifuge the 2-mL tube from step 4d at 1000 × g for 3 

minutes at 4 °C to pellet the residual nuclei. Examine the supernatant for any 

mucous aggregate material. Hold the tube at a 30° angle and, using a P200, 

transfer two 150-µL aliquots of the cell fractionation supernatant to a new 2mL 

microcentrifuge tube (a total of 300 µL). Keep the tube on ice. 

IMPORTANT! Avoid contact with or transfer of the mucous aggregate 

material. If necessary, recentrifuge the tube at 1000 × g for 3 minutes at 4 °C, 

then very carefully transfer 300 µL with a P200. 

6. Centrifuge the supernatant tube from step 3 at 16,000 × g for 15 minutes to 

pellet the Mycoplasma organisms. 

IMPORTANT! If two centrifuges are available, you can centrifuge the 

supernatant while you process the cell pellet from steps 4 through 5. 

7. Process the pellet containing Mycoplasma organisms from step 6 

immediately above: 

a. Using a P1000, carefully aspirate, then discard the supernatant. Take 

care not to touch the pellet. 

b. Transfer the 300 µL of cell fractionation lysate from step 5 above to the 

pellet from step 6, then resuspend the pellet using a P1000. Transfer 

resuspension to a new 2-mL tube. 

8. Critical step – Add 2 µL of 0.5 M EDTA and 18 µL of RNase Cocktail. 

Vortex the 2-mL tube briefly, then quick-spin the solution to bring it down 

from off of the tube wall. 

9. Incubate the tube at 56 °C for 30 minutes to digest the cellular RNA. The 

solution becomes turbid. Vortex twice during incubation. 

Note: The solution becomes clear after addition of Lysis Buffer in step 11 

below. 

10. Add 5 µL of Proteinase K. Vortex briefly, then quick-spin to bring down the 

solution from the tube wall. Incubate at 56 °C for 10 minutes. After 

incubation, set the block heater to 70 °C. 

11. Add 700 µL of Lysis Buffer. Vortex for 5 seconds at setting #7. The total 

lysate is 1 mL. 

Next step Proceed to “Extraction of Mycoplasma or Mouse Minute Virus DNA from lysate” 

on page 20. 

15 




Large-scale lysate 

preparation workflow 

16 

A large-scale lysate preparation workflow for 2- to 10-mL samples is shown 

below. For details, see page 17. 

Step 1: Place 2 to 10 mL of sample into a 50-mL tube. 

Process supernatant: keep on ice 

Step 3: Transfer the supernatant to a 

50-mL tube. Keep on ice. 

Step 5: Centrifuge at 16,000 × g for 30 min. 

Step 8a: Carefully aspirate and discard the 

supernatant. Do not disturb the 

Mycoplasma pellet. 

Step 8b: Transfer the cell fractionation 

lysate from Step 7 to the Mycoplasma 

pellet, then resuspend by pipetting using 

a P1000. Transfer to a new 2-mL tube. 

Step 9: Add 2 µL of 0.5 M EDTA and 18 

µL of RNase Cocktail. Vortex briefly, then 

quick-spin. 


Vortex twice during incubation. 

Step 11: Add 5 µL of Proteinase K. Vortex 

briefly, then quick-spin. Incubate at 56 °C 

for 10 min. 

Step 12: Add 700 µL of Lysis Buffer. Vortex 

for 5 sec at setting #7. 

Total 1 mL of lysate for DNA extraction 

Step 2: Centrifuge at 1000 × g for 5 min. 

Process cell pellet: keep on ice 

Step 4a: Add 5 mL of ice-cold 1✕ PBS. 

Use a Pasteur pipette to gently resuspend 

the cells. 

Step 4b: Centrifuge at 500 × g for 5 min. 

DIscard the supernatant. 

Step 4c: Add 550 µL of ice-cold Cell 

Fractionation Buffer to the cell pellet. Use a 

P1000 to gently resuspend the cells. Avoid 

cell lysis. 

Step 4d: Transfer to a 2-mL tube. Incubate 

on ice for up to 5 min. 

Step 6: Centrifuge at 1000 × g for 5 min at 

4 °C. Transfer 400 µL to a 2-mL tube. Avoid 

the pellet, which contains nuclei and 

mucous aggregate material. 

Step 7: Centrifuge the 2-mL tube at 

1000 × g for 3 min at 4 °C. Use a P200 to 

carefully transfer 300 µL to a 2-mL tube (to 

be processed in Step 8b). Avoid the pellet, 

which contains nuclei and mucous 

aggregate material. Recentrifuge as 

necessary. Keep on ice. 

Figure 3 Preparation of lysate from 2 to 10 mL of sample using the 

PrepSEQ Mycoplasma Nucleic Acid Extraction Kit; final sample cell 

number ≤10 8 . Critical steps are indicated in bold font. 




Prepare large-scale (2 to 10 mL) lysate (Mycoplasma detection only) 




1. Place 2 to 10 mL of the cell culture sample (final cell number ≤10 8 ) into a 

50-mL conical tube. 

2. Centrifuge the tube at 1,000 × g for 5 minutes to pellet the cells. Place the 

cell pellet on ice. 

IMPORTANT! Keep the cell pellet on ice to reduce risk of cell lysis. 

3. Transfer the supernatant to a new 50-mL conical tube. The supernatant 

contains free Mycoplasma organisms in the culture. Keep the supernatant on 

ice while working on the cell pellet; the supernatant is processed at step 5 on 

the next page. 

IMPORTANT! If two ultracentrifuges are available, you can centrifuge the 

supernatant at this step while the cell pellet is processed in the following 

steps 4 through 7. 

4. Process the cell pellet from step 2: 

a. Critical step – Gently resuspend the cell pellet in 5 mL of ice-cold 

1✕ PBS by gentle pipetting with a Pasteur pipette. 

IMPORTANT! Avoid cell lysis as much as possible. Use only ice-cold 

1✕ PBS and treat the cells very gently. Room temperature increases 

lysis of nuclei and host DNA in the final extracted DNA, and causes 

PCR inhibition. In some cases, the cell pellet is large and sticky and 

cannot be resuspended easily. Never vortex to resuspend the cells. 

b. Critical step – Centrifuge the tube at 500 × g for 5 minutes to pellet the 

cells. Completely discard the supernatant. 

c. Critical step – Add 550 µL of ice-cold Cell Fractionation Buffer to the 

cell pellet. Very gently pipette up and down several times with a P1000 

to completely resuspend the cells. 


increases lysis of the host cell nuclei leading to increased host DNA in 


17 




18 

d. Critical step – Transfer the cell suspension to a 2-mL microcentrifuge 

tube, then incubate the cell suspension on ice for 5 minutes. Do not 

exceed 5 minutes. 


increases lysis of the host cell nuclei, leading to increased host DNA in 


5. Meanwhile, centrifuge the 50-mL conical tube containing the supernatant 

from step 3 on previous page at 16,000 × g for 30 minutes to pellet the 

Mycoplasma organisms. The Mycoplasma pellet is processed in step 8. 

IMPORTANT! If two ultracentrifuges are available, you can centrifuge the 

supernatant at step 3 while you process the cell pellet from steps 4 through 7. 

6. Critical step – Centrifuge the 2-mL tube from step 4d at 1000 × g for 

5 minutes at 4 °C to pellet the nuclei. Hold the tube at a 30° angle and, using 

a P200, transfer 400 µL of the supernatant to a new 2-mL microcentrifuge 

tube. 

IMPORTANT! Avoid touching the pellet, which contains nuclei and mucous 

aggregate material that may be generated from lysis of nuclei. If necessary, 

use a P200 to perform the transfer. 

7. Critical step – Centrifuge the 2-mL tube from step 6 above at 1,000 × g for 3 

minutes at 4 °C to pellet the residual nuclei. Examine the supernatant for any 

mucous aggregate material. Hold the tube at a 30° angle and, using a P200, 

transfer two 150-µL aliquots of the cell fractionation supernatant to a new 

2-mL microcentrifuge tube (a total of 300 µL). Keep the tube on ice. 

IMPORTANT! Avoid contact with or transfer of the mucous aggregate 

material. If necessary, recentrifuge the tube at 1000 × g for 3 minutes at 4 °C, 

then very carefully transfer 300 µL with a P200. 

IMPORTANT! Keep the microcentrifuge tube on ice. 

8. Process the pellet containing Mycoplasma organisms from step 5: 

a. Using a 10-mL pipette, carefully aspirate, then discard the supernatant. 

Take care not to touch the pellet. Leave approximately 1 mL in the tube. 

Use a P1000 to carefully aspirate the remaining supernatant. 

b. Transfer the 300 µL of cell fractionation lysate from step 7 to the pellet, 

then resuspend using a P1000. Transfer to a new 2-mL tube. 

9. Critical step – Add 2 µL of 0.5 M EDTA and 18 µL of RNase Cocktail. 

Vortex the 2-mL tube briefly, then quick-spin to detach the solution from the 

tube wall. 





10. Incubate the tube at 56 °C for 30 minutes to digest the cellular RNA. The 

solution becomes turbid. Vortex twice during incubation. 

Note: The solution becomes clear after addition of Lysis Buffer in step 12 

below. 

11. Add 5 µL of Proteinase K. Vortex briefly, then quick-spin to bring down the 

solution from the tube wall. Incubate at 56 °C for 10 minutes. After 

incubation, set the block heater to 70 °C. 

12. Add 700 µL of Lysis Buffer. Vortex for 5 seconds at setting #7. The total 

lysate is 1 mL. 

Next step Proceed to “Extraction of Mycoplasma or Mouse Minute Virus DNA from lysate” 

on page 20. 

19 



Extraction of Mycoplasma or Mouse Minute Virus DNA from lysate 

Extraction of Mycoplasma or Mouse Minute Virus DNA from 

lysate 

20 

You can perform either of two DNA extraction procedures, depending on cell 

numbers and cell viability: 

Double DNA extraction – Samples with high cell numbers or low viability 

are likely to contribute proportionally high levels of DNA into the lysate. To 

ensure that the target DNA can be captured, two extractions are performed for 

these samples, and the PCR reactions are set up for both samples of extracted 

DNA. The original sample is considered positive if one of the two PCRs 

shows a positive result. 

Single DNA extraction – A single extraction is typically sufficient to capture 

all targets from samples with low cell numbers or high viability. Applied 

Biosystems recommends a single DNA extraction when you use the 

PrepSEQ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit. 

Use the following table to select the DNA extraction procedure that is 

recommended for your cell culture sample. Due to variations among cell lines and 

culture conditions, the double DNA extraction procedure is recommended for the 

initial extraction. 

Total cell number Sample type DNA extraction procedure 

≤ 106 (All samples) Single 

106 to 108 >75% viability Single 

106 to 108

Double DNA extraction 

workflow 

First DNA extraction 

Step 1a: Incubate the Magnetic Particles at 37 °C for 

10 min, then vortex the tube at setting #7 until the 

resuspension is complete. 

Steps 1b – 1c: Add 30 µL of Magnetic Particles. Add 525 µL 

of Binding Solution, then vortex for 5 min at setting #7. 

Steps 1d – 1e: Spin for 15 sec, then place the tube into a 

magnetic stand. 

Step 1f: Transfer the solution to a new 2-mL tube for use 

in step 4a. Save the tube with the magnetic particles pellet 

(first extraction) at room temp; for use in Wash DNA step 1. 

Step 1: Add 300 µL of a 3:2 mixture of 95% ethanol and 

Lysis Buffer to each extraction tube. Invert the tubes back 

and forth three times. Avoid incubation of particles in this 

wash solution for longer than 3 min. 

Steps 2 – 4: Spin for 15 sec, place the tubes into a 

magnetic stand, then aspirate and discard the liquid. 

Step 5: Add 300 µL of Wash Solution, invert the tubes back 

and forth twice, then vortex for 5 sec at setting #7. 

Steps 6 – 8: Spin for 15 sec, place the tubes into a 

magnetic stand, then aspirate and discard the liquid. 

Step 1: Add 100 µL of Elution Buffer to the first extraction 

tube and 50 µL of Elution Buffer to the second extraction 

tube. See the first elution note on page 17. 

Step 2: Vortex for 10 sec at high speed. 

Step 3: Incubate at 70 °C for 7 min. Vortex three times 

during incubation. 

Step 4: Spin for 15 sec. 




A double DNA extraction workflow is shown below. For details, see page 22. 

1 mL of sample lysate 

Bind DNA 

Wash DNA 

Elute DNA 

Second DNA extraction 

Steps 2a: Add 30 µL of Magnetic Particles to the solution 

from step 2f. 

Step 2b: Vortex for 5 min at setting #7. 

Steps 2c – 2e: Spin for 15 sec, then place the tube into a 

magnetic stand and aspirate and discard liquid. The 

Magnetic Particles pellet contains the second extraction; 

for use in Wash DNA step 1. 

Step 9: Add 300 µL of Wash Solution, invert the tubes 

back and forth twice, then vortex for 5 sec at setting #7. 

Steps 10 – 11: Spin for 15 sec, then place the tubes into a 


Step 12: Aspirate and discard the supernatant using 

a P1000 to remove residual liquid at the bottom of 

the tubes. 

Step 13: Use a P200 to aspirate residual solution. 

Step 14: Leave the tube lids open for 5 min to air dry. 

Steps 5– 6: Place the tubes into a magnetic stand. 

Transfer the eluate to a non-stick 1.5-mL tube. 

Steps 7 – 8: Spin for 3 min at top speed, then place the 

tubes into a magnetic stand. 

Step 9: Transfer the eluate to a non-stick 1.5-mL tube. 

Avoid the magnetic particles. 

Step 10: Store both eluates separately at –20 °C. 

Figure 4 Double DNA extraction using the PrepSEQ Mycoplasma Nucleic 

Acid Extraction Kit. 

21 




Double DNA extraction 

22 

In a double DNA extraction, you perform two DNA extractions. Keep the eluates 

of both extractions separate from each other (for later use in separate PCRs). 

Bind DNA 1. Perform the first DNA extraction: 

a. Incubate the Magnetic Particles at 37 °C for 10 minutes, then vortex the 

tube at setting #7 until the resuspension is complete. 

b. Add 30 µL of the Magnetic Particles to the sample. 

c. Add 525 µL of the Binding Solution to the sample. 

d. Incubate with shaking for 5 minutes at setting #7 on the vortexer to 

capture the DNA. 

e. Quick-spin the tube on a benchtop microcentrifuge for 15 seconds. This 

step spins down the solution from the tube wall and cap, and pellets 

most of the magnetic particles. 

f. Place the tube in the magnetic stand with the magnetic particles pellet 

oriented toward the magnet. 

Note: Capture of the magnetic particles with the bound DNA is 

complete after approximately 1 minute. 

g. Transfer the solution to a new 2-mL tube. Close the lid of the tube 

containing the magnetic particles. This is the first extraction. Remove 

the tube from the reagent stand and store the tube at room temperature; 

it is processed together with the second extraction at the Wash DNA 

step 1 on page 23. 

2. Perform the second DNA extraction: 

a. Add 30 µL of Magnetic Particles to the solution from step 1g above. 

(There is no need to add Binding Solution.) 

b. Incubate with shaking for 5 minutes at setting #7 on the vortexer to 

capture DNA. 

c. Quick-spin the tube on a benchtop microcentrifuge for 15 seconds. This 

step spins down the solution from the tube wall and cap, and pellets 

most of the magnetic particles. 

d. Place the tube in the magnetic stand with the magnetic particles pellet 


Note: Capture of the magnetic particles with the bound DNA is 

complete after approximately 1 minute. 

e. Aspirate and discard the supernatant carefully without disturbing the 

magnetic particles pellet. Remove the tubes from the magnetic stand. 

This is the second extraction. 




Wash DNA 1. To each of the extraction tubes from Bind DNA steps 1g and 2e, add 300 µL 

of the 95% ethanol:Lysis Buffer (3:2 v/v). Invert the tubes back and forth 

three times, then proceed immediately to the next step. 


IMPORTANT! Do not vortex. Do not incubate the samples in this wash 

solution for more than 3 minutes. 

2. Quick-spin the tubes on a benchtop microcentrifuge for 15 seconds. 

3. Place the tubes in the magnetic stand with the magnetic particles pellet 


Note: Capture of the magnetic particles with the bound DNA is complete 

after approximately 1 minute. 

4. Carefully aspirate then discard as much of the supernatant without disturbing 

the magnetic particles pellet. 

5. Add 300 µL of Wash Solution to the tubes. Invert the tubes back and forth 

twice. Vortex tubes for 5 seconds at setting #7 on the vortexer. 

Note: Flushing the magnetic particles gently with the Wash Solution helps 

detach them from the tube wall. 






8. Aspirate and discard the supernatant carefully without disturbing the 

magnetic particles pellet. 

9. Add 300 µL of Wash Solution to the tubes. Invert the tubes back and forth 

twice. Vortex the tubes for 5 seconds at setting #7 on the vortexer. 

Note: Flush the magnetic particles gently with the Wash Solution to detach 

the particles from the tube wall. 


23 




24 

11. Place tubes in the magnetic stand with the magnetic particles pellet oriented 

toward the magnet. 



12. Aspirate and discard the supernatant carefully with a P1000 without 

disturbing the magnetic particles pellet containing the bound DNA. 

13. Use a P200 to aspirate and discard the residual solution at the bottom of the 

tube. 

14. With the tube lids open, air-dry the magnetic particles pellet for 5 minutes at 

room temperature. 

Note: Ethanol in the Wash Solution decreases recovery and causes PCR 

inhibition. 

Elute DNA 1. Add 100 µL of Elution Buffer to the first extraction pellet and 50 µL of 

Elution Buffer to the second extraction pellet. 

IMPORTANT! The magnetic particles may be difficult to detach from the 

tube wall. Place the tubes in the benchtop microcentrifuge with the magnetic 

particles pellet oriented toward the center, then spin the tube for 30 seconds 

to detach the magnetic particles into the Elution Buffer. If the magnetic 

particles are difficult to resuspend, use a P200 to gently pipette up-and-down 

several times. Be careful not to let the magnetic particles pellet stick inside 

the pipette tip. 

2. Vortex the tubes for 10 seconds at high speed on the vortexer. 

3. Incubate the tubes at 70 °C for 7 minutes; vortex three times during the 

incubation to help resuspension. 




Note: Capture of the magnetic particles is complete after approximately 

1 minute. DNA is in the eluate. 

6. Transfer the liquid phase containing the eluted DNA to a new non-stick 

1.5-mL microcentrifuge tube. 

7. Centrifuge the tube for 3 minutes at top speed to pellet the residual magnetic 

particles. 





8. Place tube in the magnetic stand with the magnetic particles pellet oriented 



1.5-mL microcentrifuge tube without disturbing the magnetic particles. 

Note: Magnetic particles can be PCR inhibitory. 

10. Store both eluates separately at –20 °C. 

Note: Set up separate PCR for each sample. 

25 




Single DNA extraction 

workflow 

26 

A single DNA extraction workflow is shown below. For details, see page 27. 

Steps 3 & 4: Add 525 µL of Binding Solution, then vortex for 5 min at setting #7. 

Steps 5 & 6: Spin for 15 sec, then place the tube into a magnetic stand and aspirate and discard the liquid. 

Step 1: Add 300 µL of a 3:2 mixture of 95% ethanol and 

Lysis Buffer to each extraction tube. Invert the tubes back 

and forth three times. Avoid incubation of particles in this 

solution. 

Steps 2 – 4: Spin for 15 sec, then place the tubes into 

magnetic stand to aspirate and discard the liquid. 

Step 5: Add 300 µL of Wash Solution. Invert the tubes 

back and forth twice. Vortex for 5 sec at setting #7. 

Steps 6 – 8: Spin for 15 sec. Place the tubes into a 

magnetic stand to aspirate and discard the liquid. 

Step 1: Add 100 µL of Elution Buffer to the tube. See the 

first elution note on page 22. 

Step 2: Vortex for 10 sec at high speed. 

Step 3: Incubate at 70 °C for 7 min. Vortex three times 

during incubation. 


magnetic stand to aspirate and transfer the liquid. 

1 mL of sample lysate 

Bind DNA 

Step 1: Incubate the Magnetic Particles at 37 °C for 10 min, then vortex the tube at setting #7 until the resuspension is 

complete. 

Step 2: Add 30 µL of Magnetic Particles. 

Wash DNA 

Elute DNA 

Step 9: Add 300 µL of Wash Solution. Invert the tubes 

back and forth twice. Vortex for 5 sec at setting #7. 



Step 12: Aspirate and discard the supernatant using a 

P1000 to remove residual liquid at the bottom of the 

tubes.1 

Step 13: Use a P200 to aspirate residual solution. 

Step 14: Leave the tube lids open for 5 min to air dry. 


Steps 7 – 8: Spin for 3 min at top speed. Place the 

tubes into magnetic stand. 


Avoid the magnetic particles. 

Step 10: Store at –20 °C. 

Figure 5 Single DNA extraction using the PrepSEQ Mycoplasma Nucleic Acid 

Extraction Kit or the PrepSEQ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit. 


Single DNA extraction 




Bind DNA 1. Incubate the Magnetic Particles at 37 °C for 10 minutes, then vortex the tube 

at setting #7 until the resuspension is complete. 

2. Add 30 µL of the Magnetic Particles to the sample. 

3. Add 525 µL of the Binding Solution to the sample. 

4. Incubate with shaking for 5 minutes at setting #7 on the vortexer to capture 

DNA. 

5. Quick-spin the tube on a benchtop microcentrifuge for 15 seconds. This step 

spins down the solution from the tube wall and cap and pellets most of the 

magnetic particles. 

6. Place tube in the magnetic stand with the magnetic particles pellet oriented 






Wash DNA 1. Add 300 µL of the 3:2 mixture of Ethanol and Lysis Buffer. Invert the tube 

back and forth three times, then go immediately to the next step. 

IMPORTANT! Do not vortex. Do not incubate the samples in this wash 

solution for more than 3 minutes. 

2. Quick-spin the tube on a benchtop microcentrifuge for 15 seconds. 

3. Place the tube in the magnetic stand with the magnetic particles pellet 






27 




28 

5. Add 300 µL of Wash Solution to the tube. Invert the tubes back and forth 

twice. Vortex the tube for 5 seconds at setting #7 on the vortexer. 

Note: Flush the magnetic particles gently with the Wash Solution to detach 

the particles from the tube wall. 






8. Aspirate, then discard the supernatant carefully without disturbing the 


9. Add 300 µL of the Wash Solution to the tube. Invert the tubes back and forth 

twice. Vortex the tubes for 5 seconds at setting #7 on the vortexer. 

Note: Flushing the magnetic particles gently with the Wash Solution helps 

detach them from the tube wall. 






12. Aspirate, then discard the supernatant carefully with a P1000 without 

disturbing the magnetic particles pellet containing the bound DNA. 

13. Use a P200 to aspirate and discard the residual solution at the bottom of the 

tube. 

14. With the tube lid open, air-dry the magnetic particles pellet in the magnetic 

stand for 5 minutes at room temperature. 

IMPORTANT! Ethanol in the Wash Solution decreases recovery and causes 

PCR inhibition. 


Elute DNA 1. Add 100 µL of Elution Buffer to the tube. 




IMPORTANT! The magnetic particles may be difficult to detach from the 

tube wall. Place the tube in the benchtop microcentrifuge with the magnetic 

particles pellet oriented toward the center, then spin the tube for 30 seconds 

to detach the magnetic particles into the Elution Buffer. If the magnetic 

particles are difficult to resuspend, use a P200 to gently pipette up-and-down 

several times. Be careful not to let the magnetic particles pellet stick inside 

the pipette tip. 

2. Vortex the tube for 10 seconds at high speed. 

3. Incubate the tube at 70 °C for 7 minutes; vortex the tube three times during 

the incubation to help resuspension. 

4. Centrifuge the tube on a benchtop microcentrifuge for 15 seconds. 



Note: Complete capture of the magnetic particles occurs in approximately 

1 minute. DNA is in the eluate. 


1.5-mL microcentrifuge tube. 

7. Centrifuge the tube for 3 minutes at top speed to pellet the residual magnetic 

particles. 




1.5-mL microcentrifuge tube without disturbing the magnetic particles. 

Note: Magnetic particles can be PCR inhibitory. 

10. Store the tube at –20 °C. 

29 



Troubleshooting 

Troubleshooting 

30 

Observation Possible cause Recommended action 

Poor extraction efficiency 

(low yields) 

Ethanol is in the Wash Solution 

(step 14 on page 24 or step 14 on 

page 28). 

Magnetic particles are attached too 

tightly to the tube wall during the 

Elution (step 1 on page 24 or step 1 

on page 29). 

Magnetic particles are difficult to 

resuspend during the Elution (step 2 

on page 24 or step 2 on page 29). 

Thoroughly air-dry the magnetic particles pellet in 

the magnetic stand for 5 minutes at room 

temperature. 

Place the tube in the benchtop microcentrifuge with 

the magnetic particles pellet oriented toward the 

center. Spin the tube for 30 seconds to detach the 

magnetic particles into the Elution Buffer. 

Incubate the pellets at 70 °C for 7 minutes. Vortex 

the tubes three times during incubation to help 

resuspension. 


Product overview 

Required materials 


Appendix A 

Background and Ordering Information 

The PrepSEQ Sample Preparation Kits use magnetic particle-based separation 

technology to extract DNA from Mycoplasma cells and virus particles that are 

isolated from a variety of starting material, such as infected cell cultures or 

Mycoplasma liquid cultures. 

Kit contents The PrepSEQ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit (PN 4443789) 

contains reagents for 100 small-scale (100 µL) cell culture extractions. Kit 

components are shown in Table 1. 

Table 1 PrepSEQ 1-2-3 Mycoplasma Nucleic Acid Extraction Kit (PN 

4443789) 

Part number Description Volume Storage 

AM2286 RNase Cocktail, 1 tube 1.0 mL – 20 °C 

4400793 PrepSEQ Nucleic Acid Extraction Kit (Box 1) 

4400659 

Lysis Buffer, 2 bottles 

50 mL/bottle Room temp. 

4400789 

Binding Solution (Isopropanol), 1 empty bottle NA 

Room temp. 

4400783 

Wash Buffer Concentrate, 2 bottles 

26 mL/bottle Room temp. 

4400784 

Elution Buffer, 1 bottle 

25 mL 

Room temp. 

4400787 

Proteinase K (PK) Buffer, 1 bottle 

50 mL 

Room temp. 


4401405 Magnetic Particles, 2 tubes 1.5 mL/tube 2 to 8 °C 


4403958 Proteinase K (20 mg/mL), 1 tube 1.25 mL − 20 °C 

Note: Parts may ship separately depending on configuration and storage 

conditions. 

31



32 

The PrepSEQ Mycoplasma Nucleic Acid Extraction Kit (PN 4409733) contains 

reagents for 100 small-scale (100 to 2000 µL) or 100 large-scale (2 to 10 mL) cell 

culture extractions. Kit components are shown in Table 2. 

Note: The PrepSEQ Mycoplasma Nucleic Acid Extraction Kit (PN 4409733) 

must be ordered as part of the complete Mycoplasma kit (PN 4407876 or PN 

4409732). 

Table 2 PrepSEQ Mycoplasma Nucleic Acid Extraction Kit (PN 4409733) 

Part number Description Volume Storage 

4409733 PrepSEQ Mycoplasma Nucleic Acid Extraction Kit 

4401238 PrepSEQ Mycoplasma Nucleic Acid Extraction Kit (Module M) 

4403461 Cell Fractionation Buffer,, 1 bag containing 3 bottles (1 

bottle: PN 4405889) 

25 mL/bottle 2 to 8 °C 

4404256 RNase Cocktail, 1 box containing 2 tubes (1 

tube: 4405890 ) 

1.0 mL/tube – 20 °C 


4400659 Lysis Buffer, 2 bottles 50 mL/bottle Room temperature 

4400789 Binding Solution (Isopropanol), 1 empty bottle NA Room temperature 

4400783 Wash Buffer Concentrate, 2 bottles 26 mL/bottle Room temperature 

4400784 Elution Buffer, 1 bottle 25 mL Room temperature 

4400787 Proteinase K (PK) Buffer, 1 bottle 50 mL Room temperature 


4401405 Magnetic Particles, 2 tubes 1.5 mL/tube 2 to 8 °C 


4403958 Proteinase K (20 mg/mL), 1 tube 1.25 mL – 20 °C 

Note: Parts may ship separately depending on configuration and storage 

conditions. 


Materials not included 

in the kit 




Table 3 includes materials and equipment that are required for using (but not 

included in) the PrepSEQ Mycoplasma Nucleic Acid Extraction Kit. Unless 

otherwise indicated, many of the listed items are available from major laboratory 

suppliers (MLS). 

Table 3 Materials not included 

Item Source ‡ 

Equipment 

Three block heaters for use with 2 mL tubes: 2 set at 37 °C, and 1 MLS 

set at 56 °C 

Ice bucket MLS 

Magnetic stand, 6-tube Applied Biosystems PN AM10055 

Refrigerated benchtop microcentrifuge for 1.5- and 2-mL tubes, 

2to8°C 

MLS 

Vortex-Genie 2T Mixer VWR Scientific#14216-188 or VWR Scientific 

#14216-186 

Vortex Adapter-60, for use with Vortex-Genie Applied Biosystems PN AM10014 

Ultracentrifuge, for use with 50-mL tubes MLS 

Consumables 

Disposable gloves MLS 

Aerosol-resistant micropipette tips MLS 

Pipettors, P1000 and P200: 

MLS 

Positive-displacement 

Air-displacement 

Multichannel 

Pipettes MLS 

Tubes, conical, 50-mL Applied Biosystems PN AM12502 

Microcentrifuge tubes, non-stick RNase-free, 1.5-mL, 1 box (250 

tubes/box) 

Applied Biosystems PN AM12450 

Safe-Lock PCR clean microcentrifuge tubes, round-bottom, 2mL, 

1 bag (100 tubes/bag) 

VWR Scientific #62111-754 

Reagents 

SDS, 10% MLS 

1✕ PBS 

MLS 

IMPORTANT! Prepare fresh reagent before using kit. 

EDTA, 0.5 M MLS 

Ethanol, 95% 

IMPORTANT! Do not use denatured ethanol because it contains 

components not compatible with the protocol. 

MLS 

Isopropanol, 100% MLS 

DNase-free, sterile-filtered water MLS 

‡ For the SDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, 

refer to the SDS provided by the manufacturer, and observe all relevant precautions. 

33 




34 



This appendix covers: 

Appendix B 

Safety 

■ Chemical safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 

■ Chemical waste safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 

■ Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 

■ Chemical alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 

35

Appendix B Safety 

Chemical safety 


Chemical hazard 

warning 


guidelines 

36 

WARNING! CHEMICAL HAZARD. Before handling any chemicals, 

refer to the Safety Data Sheet (SDS) provided by the manufacturer, and 

observe all relevant precautions. 

WARNING! CHEMICAL HAZARD. All chemicals in the instrument, 

including liquid in the lines, are potentially hazardous. Always determine 

what chemicals have been used in the instrument before changing reagents 

or instrument components. Wear appropriate eyewear, protective clothing, 

and gloves when working on the instrument. 

WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles 

can crack and leak. Each 4-liter bottle should be secured in a low-density 

polyethylene safety container with the cover fastened and the handles 

locked in the upright position. Wear appropriate eyewear, clothing, and 

gloves when handling reagent and waste bottles. 

WARNING! CHEMICAL STORAGE HAZARD. Never collect or store 

waste in a glass container because of the risk of breaking or shattering. 

Reagent and waste bottles can crack and leak. Each waste bottle should be 

secured in a low-density polyethylene safety container with the cover 

fastened and the handles locked in the upright position. Wear appropriate 

eyewear, clothing, and gloves when handling reagent and waste bottles. 

To minimize the hazards of chemicals: 

Read and understand the Safety Data Sheets (SDSs) provided by the 

chemical manufacturer before you store, handle, or work with any chemicals 

or hazardous materials. (See “About SDSs” on page 37.) 

Minimize contact with chemicals. Wear appropriate personal protective 

equipment when handling chemicals (for example, safety glasses, gloves, or 

protective clothing). For additional safety guidelines, consult the SDS. 

Minimize the inhalation of chemicals. Do not leave chemical containers 

open. Use only with adequate ventilation (for example, fume hood). For 

additional safety guidelines, consult the SDS. 

Check regularly for chemical leaks or spills. If a leak or spill occurs, follow 

the manufacturer’s cleanup procedures as recommended in the SDS. 

Comply with all local, state/provincial, or national laws and regulations 

related to chemical storage, handling, and disposal. 



Chemical waste safety 

About SDSs Chemical manufacturers supply current Safety Data Sheets (SDSs) with 

shipments of hazardous chemicals to new customers. They also provide SDSs 

with the first shipment of a hazardous chemical to a customer after an SDS has 

been updated. SDSs provide the safety information you need to store, handle, 

transport, and dispose of the chemicals safely. 

Obtaining 

SDSs 


Chemical waste 

hazards 


Each time you receive a new SDS packaged with a hazardous chemical, be sure to 

replace the appropriate SDS in your files. 

The SDS for any chemical supplied by Applied Biosystems is available to you free 

24 hours a day. To obtain SDSs: 

1. Go to www.appliedbiosystems.com, click Support, then select SDS. 

2. In the Keyword Search field, enter the chemical name, product name, SDS 

part number, or other information that appears in the SDS of interest. Select 

the language of your choice, then click Search. 

3. Find the document of interest, right-click the document title, then select any 

of the following: 

Open – To view the document 

Print Target – To print the document 

Save Target As – To download a PDF version of the document to a 

destination that you select 

Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact 

the chemical manufacturer. 

CAUTION! HAZARDOUS WASTE. Refer to Safety Data Sheets (SDSs) 

and local regulations for handling and disposal. 

WARNING! CHEMICAL WASTE HAZARD. Wastes produced by 

Applied Biosystems instruments are potentially hazardous and can cause 

injury, illness, or death. 

WARNING! CHEMICAL STORAGE HAZARD. Never collect or store 

waste in a glass container because of the risk of breaking or shattering. 

Reagent and waste bottles can crack and leak. Each waste bottle should be 

secured in a low-density polyethylene safety container with the cover 

fastened and the handles locked in the upright position. Wear appropriate 

eyewear, clothing, and gloves when handling reagent and waste bottles. 

37




guidelines 

38 

To minimize the hazards of chemical waste: 

Read and understand the Safety Data Sheets (SDSs) provided by the 

manufacturers of the chemicals in the waste container before you store, 

handle, or dispose of chemical waste. 

Provide primary and secondary waste containers. (A primary waste container 

holds the immediate waste. A secondary container contains spills or leaks 

from the primary container. Both containers must be compatible with the 

waste material and meet federal, state, and local requirements for container 

storage.) 

Minimize contact with chemicals. Wear appropriate personal protective 

equipment when handling chemicals (for example, safety glasses, gloves, or 

protective clothing). For additional safety guidelines, consult the SDS. 

Minimize the inhalation of chemicals. Do not leave chemical containers 

open. Use only with adequate ventilation (for example, fume hood). For 

additional safety guidelines, consult the SDS. 

Handle chemical wastes in a fume hood. 

After emptying a waste container, seal it with the cap provided. 

Dispose of the contents of the waste tray and waste bottle in accordance with 

good laboratory practices and local, state/provincial, or national 

environmental and health regulations. 

Waste disposal If potentially hazardous waste is generated when you operate the instrument, you 

must: 

Characterize (by analysis if necessary) the waste generated by the particular 

applications, reagents, and substrates used in your laboratory. 

Ensure the health and safety of all personnel in your laboratory. 

Ensure that the instrument waste is stored, transferred, transported, and 

disposed of according to all local, state/provincial, and/or national 

regulations. 

IMPORTANT! Radioactive or biohazardous materials may require special 

handling, and disposal limitations may apply. 


Biological hazard safety 



Biological hazard safety 

General biohazard WARNING! BIOHAZARD. Biological samples such as tissues, body 

fluids, infectious agents, and blood of humans and other animals have the 

potential to transmit infectious diseases. Follow all applicable local, 

state/provincial, and/or national regulations. Wear appropriate protective 

equipment, which includes but is not limited to: protective eyewear, face 

shield, clothing/lab coat, and gloves. All work should be conducted in 

properly equipped facilities using the appropriate safety equipment (for 

example, physical containment devices). Individuals should be trained 

according to applicable regulatory and company/institution requirements 

before working with potentially infectious materials. Read and follow the 

applicable guidelines and/or regulatory requirements in the following: 

– U.S. Department of Health and Human Services guidelines published in 

Biosafety in Microbiological and Biomedical Laboratories (stock no. 

017-040-00547-4; 

http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4toc.htm ) 

– Occupational Safety and Health Standards, Bloodborne Pathogens 

(29 CFR§1910.1030; www.access.gpo.gov/ 

nara/cfr/waisidx_01/29cfr1910a_01.html) 

– Your company’s/institution’s Biosafety Program protocols for working 

with/handling potentially infectious materials. 

Additional information about biohazard guidelines is available at: 

www.cdc.gov. 

Chemical alerts 

General alerts for all 

chemicals 

Avoid contact with skin, eyes, and/or clothing. Read the SDS, and follow the 

handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 

39


Chemical alerts 

40 



Documentation 

Documentation 

For additional documentation, see “How to obtain support” on page 6. 

For information on new assays and updated product documentation, go to 

www.microseq.com. 

Document PN 

PrepSEQ Sample Preparation Kits Protocol 4401253 

PrepSEQ Sample Preparation Kits Quick Reference Card 4406304 

PrepSEQ Mycoplasma Nucleic Acid Extraction Kits Quick Reference 

Card 

MycoSEQ Mycoplasma Detection Kits Protocol (for Real-Time PCR 

Detection Kit and Mycoplasma Detection Kit) 

4406304 

4393111 

MycoSEQ Mycoplasma Detection Kits Quick Reference Card (for the 

Real-Time PCR Detection Kit and Myco Scan Mycoplasma Detection Kit) 

4393471 

ViralSEQ Mouse Minute Virus Detection Kit Protocol 4445235 

ViralSEQ Mouse Minute Virus Detection Kit Quick Reference Card 4445236 

Portable document format (PDF) versions of this guide and the documents listed 

above are available at www.appliedbiosystems.com. 

Note: To open the documentation available from the Applied Biosystems web 

site, use the Adobe ® Acrobat ® Reader ® software available at www.adobe.com. 

41

Documentation 

42 


B 

biohazardous waste, handling 39 

bold text, when to use 6 

C 

CAUTION, description 5 

chemical safety 36 

chemical waste safety 37, 38 

conventions 

bold text 6 

for describing menu commands 6 

IMPORTANTS! 6 

italic text 6 

Notes 6 

user attention words 6 

D 

DANGER, description 5 

DNA extraction 

double 20 

single 20 

double DNA extraction 

elute DNA 24 

wash DNA 23 

E 

elute DNA 24 

extraction 

double-DNA 20 

single-DNA 20 

G 

guidelines 

chemical safety 36 

chemical waste disposal 37 

chemical waste safety 38 


H 

hazards. See safety 

I 

IMPORTANT, description 5 

italic text, when to use 6 

K 

kit choice 

MMV and Mycoplasma detection 7 

Mycoplasma detection 7 

kit contents 31, 32 

L 

large-scale preprocessing 11, 13, 16 

lysate preparation 

large-scale 17 

small-scale 12 

small-scale lysate 14 

M 

Index 

magnetic particles precipitate 10 

menu commands, conventions for describing 6 

MMV detection 7 

P 

preprocessing, large-scale 11, 13, 16 

product description 31 

R 

radioactive waste, handling 38 

reagent and equipment preparation 10 

recommendation, DNA extraction procedure 20 

S 

safety 

43

Index 

biological hazards 39 

chemical 36 

chemical waste 37 

guidelines 36, 37, 38 

sample prep 

considerations 9 

Mycoplasma DNA 7 

sample size 

cell line screening and maintenance 7 

Mycoplasma detection 7 

SDSs 

about 5 

description 37 

obtaining 6, 37 

single DNA extraction 20 

bind DNA 27 

elute DNA 29 

wash DNA 27 

T 

training, information on 6 

troubleshooting 30 

U 

user attention words, described 6 

W 

WARNING, description 5 

wash DNA 23 

waste disposal, guidelines 38 

waste profiles, description 38 

44 


Part Number 4401253 Rev. C 02/2010 

Applied Biosystems 

850 Lincoln Centre Drive | Foster City, CA 94404 USA 

Phone 650.638.5800 | Toll Free 800.345.5224 

www.appliedbiosystems.com 

Technical Resources and Support 

For the latest technical resources and support information 

for all locations, please refer to our Web site at 

www.appliedbiosystems.com/support

PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit - Invitrogen

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