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ROTI®Prep DNA & RNA
20H8
Kit for simultaneous isolation of DNA and RNA from different starting
materials.
1. Introduction and product description
• Simultaneous isolation of DNA and RNA from one sample
• Preparation in the well-established mini-column system
• Fast, easy and reliable
• Extraction time of approx. 8 min (excluding lysis)
• Average puritiy DNA: 1.7-2.0, RNA: 1.8-2.1
ROTI®Prep DNA & RNA was developed to isolate both DNA and RNA separately from only one
sample.
The isolation is based on the use of two different centrifugation columns. After sample lysis, the
sample is first transferred to the first centrifugation column, which binds the DNA. The lysate is then
transferred to a second centrifugation column, to whose membrane the RNA binds. Subsequently,
DNA and RNA can be washed in parallel in the centrifugation columns and then eluted in separate
reaction vessels.
Suitable source material:
• Microbial cell cultures (max. 5x109)
• Eucaryotic cell cultures (max. 510)
• Tissue samples (max. 20 mg)
2. Product use and warranty
2.1 Intended Use
• For research use only.
• Please read the instructions for use carefully and follow the instructions accordingly. Reliability
of results cannot be guaranteed if there are any deviations in the procedure.
Information in this document is subject to change without notice. Carl Roth GmbH + Co. KG assumes
no responsibility for any errors that may appear in this document.
Carl Roth GmbH + Co. KG disclaims all warranties with respect to this document, expressed or
implied, including but not limited to those of merchantability or fitness for a particular purpose. In no
event shall Carl Roth GmbH + Co. KG be liable, whether in contract, tort, warranty, or under any
statute or on any other basis for special, incidental, indirect, punitive, multiple or consequential
damages in connection with or arising from this document, including but not limited to the use thereof.
Literature Citation: When describing a procedure for publication using this product, please refer to it
as Carl Roth’s ROTI®Prep DNA & RNA Kit
Instructions for use
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2.2 Safety Precautions
All due care and attention should be exercised in handling the materials and reagents contained in the
kit. Always wear gloves while handling these reagents and avoid any skin contact! In case of contact,
flush eyes or skin with a large amount of water immediately.
Caution:
Lysis Buffer LSR Warning H302-H304-H3012-H3014-H3018-H332
Washing Buffer WSA (conc) Warning H302-H304-H3012-H3014-H3018-H332
MSDS: the appropriate MSDS can be downloaded from our website www.carlroth.com.
3. Materials provided in this kit and storage conditions
3.1. Included Kit components
Amount
Component
Storage
6 / 30 / 125 ml
Lysis Buffer LSR
RT
6 / 30 / 2x 70 ml
Washing Buffer WSA (conc.)
RT
3 / 2x 8 / 2x 40 ml
Washing Buffer WSL (conc.)
RT
2 / 6 / 25 ml
RNase free Water
RT
2 / 6 / 30 ml
Elution Buffer EB
RT
10 / 50 / 5x 50
Mini spin columns DNA (blue)
RT
10 / 50 / 5x 50
Mini spin columns RNA (violet)
RT
20 / 2x 50 / 10x 50
1.5 ml Elution tubes
RT
40 / 4x 50 / 20x 50
2 ml Collection tubes
RT
The ROTI®Prep DNA & RNA Kit should be stored dry, at room temperature
(14-25 °C). Before every use make sure that all components have room temperature.
If there are any precipitates within the provided solutions solve these precipitates by careful warming.
This kit is stable for at least 6 months after receipt, when stored as directed.
Contents of this Kit may not be bought separately.
The user is responsible to validate the performance of the ROTI®Prep DNA & RNA Kit for any
particular use, since the performance characteristics of our kits have not been validated for any
specific application.
3.2. Required Material and Equipment not included in this kit
• Ethanol 96-99.8 %, e.g. Ethanol p.a. (e.g. 9065.1)
• TE buffer (e.g.1052.1)
• Lysozym (400 U/µl) for enzymatic lysis of bacteria (e.g. 8259.1)
Attention: Do not add bleach or acidic components to the waste after sample preparation!
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4. Application
ROTI®Prep DNA & RNA Kit is designed for isolation of high-purity genomic DNA from various source
material. Spin column-based preparation allows elution in a small volume of low-salt buffer, eliminating
time-consuming phenol-chloroform extraction or alcohol precipitation. The columns may be used either
in micro-centrifuges or on vacuum manifolds.
The kit allows the extraction of up to 40 µg DNA and 60 µg RNA per preparation from microbial cell
cultures (max. 1x109), fungi and eukaryotic cell cultures (max. 5x106), up to 20 mg Tissue samples.
During lysis, the sample has to be mixed carefully and, if possible, constantly. Reduced movement of
the lysis mix will reduce the lysis efficiency, and, subsequently, the recovery rate of gDNA. We
recommend using a shaking platform or thermomixer in order to keep the sample at constant
movement.
Store the extracted DNA at +4 °C and the extracted RNA at -20°C. For long time storage placing at
-20 °C (DNA) or -80 °C (RNA) is recommended.
For centrifugation we recommend a standard microcentrifuge. Centrifugation steps should be carried
out at room temperature.
Avoid repeated freeze&thaw cycles for the tissue to be extracted.
4.1 General Comments on Handling of RNA
RNA is far less stable than DNA. It is very sensitive to degradation by endogenous RNases in the
biological material and exogenous RNases which are permanently present everywhere in the lab. In
order to achieve satisfactory qualitative and quantitative results in RNA preparations, contaminations
with exogenous RNases have to be reduced to a minimum in accordance with the following
recommendations:
• Clear the bench top first using RNase AWAY® (A998.1) or ROTI®Nucleic Acid-free (HP69.1).
• Always wear latex or vinyl gloves while handling reagents and RNA samples in order to
prevent RNase contaminations from surface of the skin or from dusty laboratory equipment.
• Use only sterile, disposable polypropylene tubes throughout the procedure (these tubes are
generally RNase-free.)
• Autoclaving will not inactivate RNases.
• Non-disposable plastic ware should be treated before use to ensure that it is RNase-free.
Plastic ware should be thoroughly rinsed with RNase AWAY® (A998.1) or ROTI®Nucleic
Acid-free (HP69.1), followed by RNase-free water.
• All glassware should be treated before use in order to ensure that it is RNase-free. Glassware
should be cleaned with RNase AWAY® (A998.1) or ROTI®Nucleic Acid-free (HP69.1),
thoroughly rinsed with RNase-free water, and oven baked at 240 °C for four or more hours
before use. Oven baking in-activates RNases and ensures that no other nucleic acids (such
as plasmid DNA) are present on the surface of the glassware.
• Reduce preparation time as much as possible.
• Change gloves frequently and keep tubes closed.
• Keep isolated RNA on ice.
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• Electrophoresis tanks should be cleaned with RNase AWAY® (A998.1) or ROTI®Nucleic
Acid-free (HP69.1), followed by RNase-free water, rinsed with ethanol and finally allowed to
dry.
• All buffers have to be prepared with DEPC-treated RNase-free double-distilled water.
• Avoid handling bacterial cultures, cell cultures or other biological sources of RNases in the
same lab where the RNA purification will be performed.
• Do not use equipment, glassware and plastic ware employed for other applications which
might introduce RNase contaminations in the RNA isolation.
4.2. Before start, be sure to…
• Add Ethanol (96-99.8 %, not incl. in this kit) to the Washing Buffer WSA (conc.) as follows:
20H8.1 (10 Preps): 6 ml (12 ml final vol.)
20H8.2 (50 Preps): 30 ml (60 ml final vol.)
20H8.3 (250 Preps): 70 ml to each bottle (140 ml final vol.)
Mix thoroughly and keep the bottle always firmly closed!
• Add Ethanol (96-99.8 %, not incl. in this kit) to the Washing Solution WSL (conc.) as follows:
20H8.1 (10 Preps): 12 ml (15 ml final vol.)
20H8.2 (50 Preps): 32 ml to each bottle (2x 40 ml final vol.)
20H8.3 (250 Preps): 160 ml to each bottle (2x 200 ml final vol.)
Mix thoroughly and keep the bottle always firmly closed!
• Heat one thermal mixer or water bath to 56 or 60 °C.
• Pre-fill the needed amount of RNase free water or Elution Buffer into a 2.0 ml reaction tube and
incubate at 60 °C until the elution step.
• For Lysis steps we recommend using a shaking platform (thermal mixer, water bath or another
rocking platform) for a continuous shaking of the sample. Alternatively, vortex the sample 3–4
times during the incubation. No shaking will reduce the lysis efficiency.
4.3. Workflow
The workflow has been provided on a separate page, in order to use a copy for in-process protocol or
filing in your personal lab files.
• DNA isolation from bacterial cell cultures
• DNA isolation from eucaryotic cell cultures
p. 4
p. 5
• DNA isolation from tissue samples
p. 6
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4.3.1 DNA isolation from bacterial cell cultures
Step (RT = room temperature)
done
1. Collection of bacterial cells
Transfer the bacterial cell culture into a 2.0 ml reaction tube
Pellet cells by centrifugation at 5000 g (or 7000 rpm) for 5 mins and discard the supernatant
Resuspend the bacterial cell pellet in 100 µl TE-Buffer.
2.1 Lysis of bacterial cells
Add 20 µl Lysozyme (10mg/ml, 400 U/µl) and incubate at 37 °C for 30 minutes under continuous shaking
(obligatory step for gram-positive bacteria, optional step for gram-negative bacteria). For Staphylocuccus 10
µl Lysostaphin (0.4 U/µl) are recommended
Add 450 µl Lysis Buffer LSR to the sample and vortex the sample shortly
Incubate the sample for 3 minutes at RT
Centrifuge at max. speed for 1 min to spin down the unlysed material. Do not discard the supernatant
2. Column Loading
Place each Spin Column DNA (blue ring) into a 2 ml collection tube
Apply the supernatant to the spin column.
Centrifuge at 11.000 g (or 13.000 rpm) for 2 min/RT.
Note: Do not discard the flowthrough as it contains the RNA. Also keep the spin column as is contains the
DNA
Add an equal volume of 70 % ethanol (p.a.) to the flow-through and mix by carefully pipetting up and down.
Place each Spin Column RNA (violet ring) into a fresh 2 ml collection tube
Transfer the flowthrough/ethanol mix (max. 650 µl) to the spin column RNA
Centrifuge at 11.000 g (or 13.000 rpm) for 2 min/RT and discard the flowthrough and place the spin column
back into the collection tube spin.
Repeat the last centrifugation step if there is any residual sample.
3. Column Washing (DNA and RNA in parallel)
Place the Spin Column DNA (blue ring) into a new 2 ml collection tube. It is washed in parallel with the
Spin column RNA (violet).
Add 500 µl of Washing Buffer WSA to the Spin Column DNA and to Spin Column RNA
Centrifuge both vessels at 11.000 g (or 13.000 rpm) for 1 min/RT, discard the flow-through and reuse the
collection tube
Place the Spin Column back into the 2 ml collection tube
Add 700 µl of Washing Buffer WST to the Spin Column
Centrifuge at 6000 g (or 8000 rpm) for 1 min/RT, discard the flow-through and reuse the collection tube
Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 2mins/RT in order to remove residual
ethanol.
4. Elution
Place each Spin Column into a clean 1.5 ml elution tube
Add 30-80 µl RNase free Water to spin column RNA (violet) and 100 µl Elution Buffer to spin column DNA
(blue ring) the centre of the membrane.
Incubate for 2 min at room temperature
Centrifuge at 11.000 g (or 13.000 rpm) for 1 min/RT to elute DNA
Repeat the elution step (by loading the eluted DNA again on the spin column) to increase the yield of
extracted DNA
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4.3.2 DNA isolation from eucaryotic cell cultures
Step (RT = room temperature)
done
1. Collection of cells
Transfer up to 5 x 106 eukaryotic cells into a 2.0 ml reaction tube. Pellet cells by centrifugation at 5000 g (or
7000 rpm) for 5 mins and discard the supernatant.
2.1 Lysis of cells cells
Add 400 µl Lysis Buffer LSR to the sample and vortex the sample shortly
Incubate the sample for 2 min at RT. Resuspend the cells completely by pipetting up and down. Incubate the
sample for further 3 min at RT.
Note: In order to obtain maximum yield of DNA and RNA the cells should be completely lysed to proceed
Centrifuge at max. speed for 1 min to spin down the unlysed material. Do not discard the supernatant
2. Column Loading
Place each Spin Column DNA (blue ring) into a 2 ml collection tube
Apply the supernatant to the spin column.
Centrifuge at 11.000 g (or 13.000 rpm) for 2 min/RT.
Note: Do not discard the flowthrough as it contains the RNA. Also keep the spin column as is contains the
DNA
Add an equal volume of 70 % ethanol (p.a.) to the flow-through and mix by carefully pipetting up and down.
Place each Spin Column RNA (violet ring) into a fresh 2 ml collection tube
Transfer the flowthrough/ethanol mix (max. 650 µl) to the spin column RNA
Centrifuge at 11.000 g (or 13.000 rpm) for 2 min/RT and discard the flowthrough and place the spin column
back into the collection tube spin.
Repeat the last centrifugation step if there is any residual sample.
3. Column Washing
Place the Spin Column DNA (blue ring) into a new 2 ml collection tube. It is washed in parallel with the
Spin column RNA (violet).
Add 500 µl of Washing Buffer WSA to the Spin Column DNA and to Spin Column RNA
Centrifuge both vessels at 11.000 g (or 13.000 rpm) for 1 min/RT, discard the flow-through and reuse the
collection tube
Place the Spin Column back into the 2 ml collection tube
Add 700 µl of Washing Buffer WSL to the Spin Column
Centrifuge at 6000 g (or 8000 rpm) for 1 min/RT, discard the flow-through and reuse the collection tube
Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 2mins/RT in order to remove residual
ethanol.
4. Elution
Place each Spin Column into a clean 1.5 ml elution tube
Add 30-80 µl RNase free Water to spin column RNA (violet) and 100 µl Elution Buffer to spin column DNA
(blue ring) the centre of the membrane.
Incubate for 2 min at room temperature
Centrifuge at 11.000 g (or 13.000 rpm) for 1 min/RT to elute DNA
Repeat the elution step (by loading the eluted DNA again on the spin column) to increase the yield of
extracted DNA
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4.3.3 DNA isolation from tissue samples
Step (RT = room temperature)
done
2.1 Lysis of cells cells
Cut max. 20 mg of tissue/biopsie sample into small pieces and homogenize the sample. Homogenization can either be
performed by:
• Using mortar pestle (grind with liquid nitrogen and avoid thawing, then transfer the sample to a reaction tube and
add 450 µl Lysis Buffer LSR
• Using a SampleLyse Tube e.g. 1YKA.1 and a speed mill. If you are doing this you can add the Lysis Buffer LSR
(450 µl) directly to the sample in the lysis tube. Homogenize the sample in a rotor stator mill.
Note: In order to obtain maximum yield of DNA and RNA the cells should be completely lysed to proceed
Centrifuge at max. speed for 1 min to spin down the unlysed material. Do not discard the supernatant
2. Column Loading
Place each Spin Column DNA (blue ring) into a 2 ml collection tube
Apply the supernatant to the spin column.
Centrifuge at 11.000 g (or 13.000 rpm) for 2 min/RT.
Note: Do not discard the flowthrough as it contains the RNA. Also keep the spin column as is contains the
DNA
Add an equal volume of 70 % ethanol (p.a.) to the flow-through and mix by carefully pipetting up and down.
Place each Spin Column RNA (violet ring) into a fresh 2 ml collection tube
Transfer the flowthrough/ethanol mix (max. 650 µl) to the spin column RNA
Centrifuge at 11.000 g (or 13.000 rpm) for 2 min/RT and discard the flowthrough and place the spin column
back into the collection tube spin.
Repeat the last centrifugation step if there is any residual sample.
3. Column Washing
Place the Spin Column DNA (blue ring) into a new 2 ml collection tube. It is washed in parallel with the
Spin column RNA (violet).
Add 500 µl of Washing Buffer WSA to the Spin Column DNA and to Spin Column RNA
Centrifuge both vessels at 11.000 g (or 13.000 rpm) for 1 min/RT, discard the flow-through and reuse the
collection tube
Place the Spin Column back into the 2 ml collection tube
Add 700 µl of Washing Buffer WSL to the Spin Column
Centrifuge at 6000 g (or 8000 rpm) for 1 min/RT, discard the flow-through and reuse the collection tube
Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 2mins/RT in order to remove residual
ethanol.
4. Elution
Place each Spin Column into a clean 1.5 ml elution tube
Add 30-80 µl RNase free Water to spin column RNA (violet) and 100 µl Elution Buffer to spin column DNA
(blue ring) the centre of the membrane.
Incubate for 2 min at room temperature
Centrifuge at 11.000 g (or 13.000 rpm) for 1 min/RT to elute DNA
Repeat the elution step (by loading the eluted DNA again on the spin column) to increase the yield of
extracted DNA
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5. Trouble Shooting
Problem / probable cause
Comments and suggestions
1. Clogged spin filter
Insufficient lysis and/or too much starting
material
Increase lysis time.
Increase centrifugation speed.
After lysis centrifuge the lysate to pellet unlysed
material.
Check storage conditions and usage of Proteinase K.
Optionally replace Proteinase K by a fresh lot.
Reduce amount of starting material.
Overload of filters reduces yield.
2. Low recovery
Insufficient lysis
See above
Insufficient mixing with Binding Buffer BSN
Mix sample very well with Binding Buffer BSN by
pipetting or by vortexing prior to transfer of the sample
onto the Spin Filter
Low amount of recovered gDNA.
Add the Elution Buffer directly onto the centre of the
Spin Column.
Prolong the incubation time with Elution Buffer.
Increase volume of Elution Buffer used or repeat elution
step.
Low concentration of eluted gDNA
Reduce the volume of Elution Buffer used. Optionally:
prolong elution / incubation time.
3. Degraded or sheared DNA
Incorrect storage of starting material.
Make sure that the starting material is frozen
immediately in liquid N2 or in minimum at
-20 °C, and is stored continuously at -80 °C!
Avoid repeated freezing and thawing of the starting
material.
Old material
Use fresh material. Check and improve storage
conditions.
4. Poor quality of RNA
Ethanol carry-over during elution
Increase time for removing ethanol
Ensure that both Washing buffers have room
temperature.
Check up Washing Buffers for salt precipitates.
If there are any precipitate dissolve these precipitate by
carefully warming.
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Ordering information:
(for detailed kit content see Table under 1.)
ROTI®Prep DNA & RNA 10 preps 20H8.1
ROTI®Prep DNA & RNA 50 preps 20H8.2
ROTI®Prep DNA & RNA 250 preps 20H8.3
Carl Roth GmbH + Co. KG
Schoemperlenstraße 3-5 • 76185 Karlsruhe
P.O. Box 100121 • 76231 Karlsruhe
Phone: +49 (0) 721/ 5606-0
Fax: +49 (0) 721/ 5606-149
info@carlroth.com • www.carlroth.com LHa 03/2023
The comp any is a limited partnership with headquarters in Karlsruhe, r eg. court
Mannheim HRA 100055. Roth C hemie Gmb H, with headquarters in Ka rlsruhe, reg.
court Mannheim HRB 100428, is the persona lly liabl e partner. Man aging Director:
André Houdelet. Sales tax identification n umber: DE 1 43621073.
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