INVITEK
Molecular
User manual
RTP Bacteria DNA M ini Kit
for purification of genomic DNA from bacteria species
1033200x00 Invitek Molecular GmbH, D-13125 Berlin
Instruction for the RTP® Bacteria DNA Mini Kit
The RTP® Bacteria DNA Mini Kit allows rapid and efficient isolation of high-quality,
chromosomal bacterial DNA from bacterial species, tissue samples, paraffin embedded tissue, urine, paper points, swabs and water using the RTP® technology. The purified DNA can be used for in-vitro diagnostic analysis.
The kit is neither validated for the isolation of genomic DNA from cell free body fluids, like cerebrospinal fluid, synovial fluid or for DNA from stool sample, parasites or the purification of total RNA.
The application of the kit for isolation and purification of viral DNA has not been evaluated.
CC
IVD
Compliance with EU Directive 98/79/EC on in vitro medical devices.
Not for in-vitro diagnostic use in countries where the EU Directive 98/79/EC on in vitro medical devices is not recognized.
Trademarks: Invisorb®, RTP®, InviMag®, Eppendorf®. Registered marks, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.
The lnvisorb®technology is covered by patents and patent applications: US 6,110363, US 6,043,354, US 6,037,465, EP 0880535, WO 9728171, WO 9534569, EP 0765335, DE 19506887, DE 10041825.2, WO 0034463.
Invisorb®, RTP® and InviMag® are registered trademarks of Invitek Molecular GmbH.
The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG.
©2019 Invitek Molecular, all rights reserved.
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RTP® Bacteria DNA Mini Kit 0419
Contents
Kit contents of RTP® Bacteria DNA Mini Kit 3
Symbols 4
Storage 4
Quality control 4
Intended use 5
Product use limitations 5
Safety information 6
Product characteristic of RTP® Bacteria DNA Mini Kit 7
Principle and procedure 8
Sampling and storage of starting material 8
Sample preparation 9
Yield and quality of genomic DNA 9
Important notes 10
Preparing reagents and buffers 10
Equipment and reagents to be supplied by user 11
Important indication 11
Scheme of RTP® Bacteria DNA Mini Kit 12 Protocols
Protocol 1: Isolation of bacterial DNA from swabs 13
Protocol 2: Isolation of DNA from bacteria pellets (1 x 109 bacteria cells) 14
Protocol 3: Isolation of bacterial DNA from Listeria ssp. from food samples 15
Protocol 4: Isolation of DNA from periodonthopathogenic bacteria from 16 paper points
Protocol 5: Isolation of bacterial DNA from tissue biopsies 17 Protocol 6: Isolation of bacterial DNA from paraffin embedded tissue sample 18
Protocol 7: Isolation of bacterial DNA from urine sample 19
Protocol 8: Isolation of bacterial DNA from water sample (big volume) 20
Troubleshooting 21
Appendix 22
General notes on handling DNA 22
Ordering information 23
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RTP® Bacteria DNA Mini Kit 0419
Kit contents of RTP® Bacteria DNA Mini Kit
	5 DNA-extractions	50 DNA-extractions	250 DNA-extractions
Catalogue No.	1033200100	1033200200	1033200300
Extraction Tube	5	50	5x50
Resuspension Buffer R	2x2 ml	30 ml	150 ml
Binding Buffer B6	3x 1 ml (ready to use)	9 ml (final volume 30 ml)	36 ml (final volume 120 ml)
Elution Buffer	2 ml	30 ml	120 ml
Wash Buffer 1	15 ml (ready to use)	30 ml (final volume 60 ml)	80 ml (final volume 160 ml)
Wash Buffer II	15 ml (ready to use)	18 ml (final volume 60 ml)	60 ml (final volume 200 ml)
RTA Spin Filter Set	5	50	5x50
RTA Receiver Tube	5	50	5x50
1.5 ml Receiver Tubes	5	50	5x50
Manual	1	1	1
Initial steps		Add 21 ml 99.7% Isopropanol to the Binding Buffer B6. Mix by intensive shaking by inverting for 1 min. Shortly before use mix by inverting several times. Add 30 ml of 96%-100%ethanol to the bottle Wash Buffer I, mix shortly and keep the bottle always firmly closed! Add 42 ml of 96%-100%ethanolto the bottle Wash Buffer II, mix shortly and keep the bottle always firmly closed!	Add 84 ml 99.7% Isopropanol to the Binding Buffer B6. Mix by intensive shaking by inverting for 1 min. Shortly before use mix by inverting several times. Add 80 ml of 96% -100 % ethanolto the bottle Wash Buffer I, mix shortly and keep the bottle always firmly closed! Add 140 ml of 96% -100% ethanolto each bottle Wash Buffer II, mix shortly and keep the bottle always firmly closed!
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RTP® Bacteria DNA Mini Kit 0419
Symbols
Manufacturer
[lÖtI    Lot number Attention: Do not combine components of different kits,
unless the lot numbers are identical I
ref|   Catalogue number ^    Expiry date
Qi]   Consult operating instructions jj£    Temperature limitation
Do not reuse |p    Humidity limitation
Storage
All buffers and kit contents of the RTP® Bacteria DNA Mini Kit including the Extraction Tubes (incl. Lysis Buffer, Proteinase K, Carrier RNA, Lysozyme and Internal Control DNA) should be stored at room temperature and are stable for at least 12 months. Store the Kit especially the Extraction Tubes in a dry environment, the Extraction Tubes must be protected from humidity.
Room temperature (RT) is defined as range from 15-30°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 warming carefully (up to 30°C).
Wash Buffers charged with ethanol should be appropriately sealed and stored at room temperature.
Binding Buffer charged with isopropanol should be appropriately sealed and stored at room temperature.
Quality control and product warranty
Invitek Molecular warrants the correct function of the RTP® Bacteria DNA Mini Kit for applications as described in this manual. Purchaser must determine the suitability of the Product for its particular use. Should any Product fail to perform the applications as described in the manual, Invitek Molecular will check the lot and if Invitek Molecular investigates a problem in the lot, Invitek Molecular will replace the Product free of charge. Invitek Molecular reserves the right to change, alter, or modify any Product to enhance its performance and design at any time. In accordance with Invitek Molecular's EN ISO 13485 certified Quality Management System the performance of all components of the RTP® Bacteria DNA Mini Kit have been tested separately against predetermined specifications routinely on lot-to-lot to ensure consistent product quality. If you have any questions or problems regarding any aspects of RTP® Bacteria DNA Mini Kit or other Invitek Molecular products, please do not hesitate to contact us. A copy of Invitek Molecular's terms and conditions can be obtained upon request or are presented at the Invitek Molecular webpage www.invitek-molecular.com.
For technical support or further information please contact:
from Germany +49-(0)30-9489-2901/ 2910
from abroad +49-(0)30-9489-2907
or contact your local distributor.
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RTP® Bacteria DNA Mini Kit 0419
Intended use
The RTP® Bacteria DNA Mini Kit allows rapid and efficient isolation of high quality DNA from bacterial species, food and tissue samples, paraffin embedded tissue, urine, paper points, swabs and water using the RTP® technology.
This kit technology yields bacterial DNA from different human samples that is free of proteins, nucleases and other impurities and is ready to use for different downstream applications, such as PCR, quantitative PCR and others. The purified DNA can be used for in-vitro diagnostic analysis only.
THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY, SUCH AS TECHNICIANS, PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES. It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification. Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings.
To minimize irregularities in diagnostic results, adequate controls for downstream applications should be used.
The kit is in compliance with EU Directive 98/79/EC on in vitro medical devices. But it is not for in-vitro diagnostic use in countries where the EU Directive 98/79/EC on in vitro medical devices is not recognized.
Product use limitation
The kit is neither validated for the isolation of genomic DNA from cell free body fluids, like cerebrospinal fluid, synovial fluid or for DNA from stool sample, parasites or the purification of total RNA.
The application of the kit for isolation and purification of viral DNA has not been evaluated. The included chemicals are only useable once.
Differing of starting material or flow trace may lead to inoperability; therefore, neither a warranty nor guarantee in this case will be given, neither implied nor express.
The user is responsible to validate the performance of the Invitek Molecular Product for any particular use. Invitek Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications.
Invitek Molecular products may be used e.g. in clinical diagnostic laboratory systems under following conditions:
o   If used in the US, based on the condition that the complete diagnostic system of the laboratory has been validated pursuant to CLIA' 88 regulations.
o   For other countries based on the condition that the laboratory has been validated pursuant to equivalents according to the respective legal basis.
All Products sold by Invitek Molecular are subject to extensive quality control procedures (according to EN ISO 13485) and are warranted to perform as described herein. Any problems, incidents or defects shall be reported to Invitek Molecular immediately upon detection thereof.
The chemicals and the plastic parts are for laboratory use only; they must be stored in the laboratory and must not be used for purposes other than intended.
The Product with its contents is unfit for consumption.
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RTP® Bacteria DNA Mini Kit 0419
Safety information
When and while working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles!
Avoid skin contact! Adhere to the legal requirements for working with biological material! For more information, please consult the appropriate material safety data sheets (MSDS). These are available online in convenient and compact PDF format at www.invitek-molecular.com for each Invitek Molecular Product and its components. If buffer bottles are damaged or leaking, WEAR GLOVES, AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries.
Invitek Molecular has not tested the liquid waste generated by the RTP® Bacteria DNA Mini Kit procedures for residual infectious materials. Contamination of the liquid waste with residual infectious materials is highly unlikely, but cannot be excluded completely. Therefore, liquid waste must be considered infectious and be handled and discarded according to local safety regulations.
European Community risk and safety phrases for the components of the RTP® Bacteria DNA Mini Kit to which they apply is listed are listed below as follows:
Wash Buffer I
<!>
\r Warning
H302-H412-P280-P305-P351-P338-P273-EUH032
Extraction Tubes
<!><|>
Warning
H302-H315-H319-H335-H411-P280-P305+P351+P338-EUH208
Wash Buffer I (ready to use)
Danger H225-P403+P233
Wash Buffer II (ready to use)
Danger H225-P403+P233
Binding Buffer B6 (ready to use)
\r      \r Danger H225-H319-H336-P210-233-P305+P351+P338
H225: Highly flammable liquid and vapour.
H302: Harmful if swallowed.
H315: Causes skin irritation.
H319: Causes serious eye irritation.
H332: Harmful if inhaled.
H335: May cause respiratory irritation.
H336: May cause drowsiness or dizziness.
H411: Toxic to aquatic life with long lasting effects.
H412: Harmful to aquatic life with long lasting effects.
P210: Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.
P233: Keep container tightly closed.P280: Wear protective gloves/protective clothing/eye protection/face protection.
P273: Avoid release to the environmentP305+P351+P338: IF IN EYES: Rinse cautiously with water for several
minutes. Remove contact lenses, if
present and easy to do. Continue rinsing.
P403+P233: Store in a well-ventilated place. Keep container tightly closed.
EUH208: Contains Proteinase, Tritirachium album-Serine. May produce an allergic reaction.
EUH032: Contact with acids liberates very toxic gas.
Emergency medical information can be obtained 24 hours a day from infotrac:
outside of USA: 1 - 352 - 323 - 3500 in USA : 1 - 800 - 535 - 5053
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RTP® Bacteria DNA Mini Kit 0419
Product characteristic of the RTP® Bacteria DNA Mini Kit
Starting material	Yield	Time	Ratio
max. 109 bacteria, 1-10 mg tissue, biopsy material, paraffin embedded tissue, swabs 15 - 50 ml urine, paper points	depends on the starting material	25 min (without lysis time)	A260 : A 280 1.7-2.0
The RTP® Bacteria DNA Mini Kit allows rapid and efficient isolation of high quality genomic DNA from bacteria species (up to 109 bacteria cells) included in different kinds of starting materials (bacteria pellets, paper points).
The kit uses the patented RTP® technology, whose special feature is the utilization of an Extraction Tube containing pre-formulated solid lysis reagent, lytic enzyme, Proteinase K, carrier nucleic acid and a precisely calibrated amount of an internal DNA Extraction Control.
The internal control is a convenient tool for the assessment and monitoring of extraction efficiency and PCR amplification as well, for the quality of the purified nucleic acid, and for the exclusion of false negative results.
Using the RTP® Bacteria DNA Mini Kit, all types of samples are transferred into the Extraction Tubes together with a specially designed Resuspension Buffer. The prefilled buffer and enzymes lyse the samples, stabilize nucleic acid and enhance the selective DNA adsorption to the membrane. The membranes are efficiently washed before the nucleic acid is eluted. In addition to the rigorous lysis procedure, simple pre-treating steps have been introduced, ideally for purification of genomic DNA, especially from Gram-positive bacteria of different sources. High extraction efficiency and detection sensitivities will be realized. No phenol/chloroform extraction is required. The "hands-on time" necessary for the whole procedure is reduced to a minimum. All kit components can be stored at room temperature.
The procedures require minimal interaction by the user, allowing safe handling of potentially infectious samples.
Due to the high purity, the isolated total DNA is ready to use for a broad panel of downstream applications (see below) or can be stored at -80°C for subsequent use.
o PCR
o   Real-time PCR (quantitative RT-PCR, like TaqMan und LightCycler technology) o   Mircoarray application o RFLP-Analysis
Invitek Molecular offers the InviMag® Bacteria DNA Mini Kit / KFmL for DNA isolation using magnetic beads.
For further information please contact: phone: +49 (0) 30 9489 2901, 2910 in Germany and from foreign countries phone: +49 (0) 30 9489 2907 or your local distributor.
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RTP® Bacteria DNA Mini Kit 0419
Principle and procedure
The RTP® Bacteria DNA Mini Kit procedure comprises following steps: o   lysis at different temperatures
o   binding the nucleic acids in the lysate to the membrane of a RTA Spin Filter o  washing of the membrane and elimination of contaminants and ethanol o  elution of nucleic acid
This manual contains 8 protocols.
Sampling and storage of starting material
Pathogens (Listeria ssp.) in food material:
For the detection of bacteria (Listeria) in foods they must be enriched and cultivated following the EU regulations and § 35 of the food law. An aliquot of the culture will only be used and the bacteria will be pelleted after cultivation. Best results are obtained with fresh material or material that has been immediately frozen and stored at -20°C or -80°C. Repeated freezing and thawing of stored samples should be avoided, since this leads to reduced DNA size.
Bacterial cultures:
Bacterial cultures grow in the presence of a selective agent such as an antibiotic. The yield and quality of DNA may depend on factors such host strain, inoculation, antibiotic, and type of culture medium. The bacteria will be pelleted after cultivation. Best results are obtained with fresh material or material that has been immediately frozen and stored at -20°C or -80°C. Repeated freezing and thawing of stored samples should be avoided, since this leads to reduced DNA size.
Paper Points:
These can be stored at room temperature up to 6 hours. For long term storage, we recommend dry storage at 2-8°C.
Biopsy material / tissue / paraffin embedded tissue:
Best results are obtained with fresh material or material that has been immediately frozen and stored at -20°C or -80°C. Repeated freezing and thawing of stored samples should be avoided, since this leads to reduced DNA size. Use of poor quality starting material influences yield of purified DNA. The thawing process could proceed, e.g. in the Extraction Tube.
Tissue/ Biopsy Material:
Best results are obtained with fresh material or material that has been immediately frozen and stored at -20°C or -80°C. Repeated freezing and thawing of stored samples should be avoided, since this leads to reduced DNA size. Use of poor quality starting material also leads to reduced length and yield of purified DNA. The amount of purified DNA from max. 10 mg tissue sample depends on the nature of starting material.
Urine:
The bacteria must be pelleted and the supernatant completely removed (urea contaminations can inhibit PCR reactions). Best results are obtained with fresh peletted material or bacteria pellets that has been immediately frozen and stored at -20°C or -80°C. Freezing and thawing of stored samples should be avoided, since this leads to reduced DNA size. The amount of purified DNA from max. 15-50 ml urine, depends from the included bacteria titre.
Swabs, Saliva
The protocol works with fresh saliva, prepared swabs as well as with dried swabs. Please note, that stored and dried swab sample often characterized by isolation of apoptotic DNA (visible on agarose gel as typical apoptotic DNA banding pattern).The protocol has not been validated for isolation of DNA from swabs which are stored under special storage buffers of other provider.
Invitek Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified.
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RTP® Bacteria DNA Mini Kit 0419
Sample preparation
Bacteria must be cultivated under special conditions and an aliquot of the bacteria suspension will be used to win a bacteria pellet by centrifugation at high speed for 5 min. The supernatant will be removed.
Lysis
Samples are lysed under non-chaotropic conditions at different, elevated temperature and continuously shaking. Lysis is performed in the presence of Lysozym to break the cell wall of the bacteria, a Lysis Buffer and Proteinase K to digest the proteins. All is provided prefilled in the Extraction Tube. Unlysed sample parts should be removed before the binding step.
Binding genomic DNA
By adding Binding Buffer B6 to the lysate, optimal binding condition will be adjusted. Each lysate is then applied to a RTA Spin filter and genomic DNA is adsorbed onto membrane as the lysate is drawn through by centrifugal force.
Removing residual contaminants
Contaminants are efficiently washed away using Wash Buffers, while the bacterial, genomic DNA remains bound to the membrane.
Elution of pure genomic DNA
Genomic DNA is eluted from the spin filter using 50 - 200 ul pre-warmed Elution Buffer or water. Eluting twice each with 100 ul leads to little increase of DNA yield. Usage small elution volumes may raise DNA concentration. Elution volumes should be at least 50 ul. The eluted DNA is ready for use in different downstream applications.
Yield and quality of genomic DNA
The amount of purified DNA using the RTP® Bacteria DNA Mini Kit procedure depends on the sample type and the number of bacteria cells in the sample (depending from the source, e.g. patients age and health situation, transport conditions, storage, and age of the sample).
For most samples, a single elution with 200 ul Elution Buffer is sufficient.
Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction. Eluates derived by this kit will contain Carrier-RNA, which will greatly exceed the amount of the isolated NA.
Yields of viral nucleic acids isolated from biological samples are usually low concentrated and therefore almost impossible to determine photometrically*
* Keep in mind that the Carrier-RNA (5 ug per 200 ul sample) will account for most of the present RNA.
The kit is suitable for downstream analysis with NAT techniques, for examples qPCR, RT qPCR, LAMP, LCR. Diagnostic assays should be performed according to the manufacturer's instructions.
Quantitative RT-PCR is recommended for determination of bacterial DNA yield.
* In Gel Electrophoresis and in Capillary Electrophoresis, DNA extracted with the provided kit looks like degraded cause the kit contains Carrier RNA, this is poly A RNA in fragments of 100 up to 1000 bases. The kit is not dedicated for applications using this kind of analysis.
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RTP® Bacteria DNA Mini Kit 0419
Important notes
Important points before starting a protocol
Immediately upon receipt of the Product, inspect the Product and its components as well as the package for any apparent damages, correct quantities and quality. If there are any unconformities you have to notify Invitek Molecular in writing with immediate effect upon inspection thereof. If buffer bottles are damaged, contact the Invitek Molecular Technical Services or your local distributor. In case of liquid spillage, refer to "Safety Information" (see page 7). Do not use damaged kit components, since their use may lead to poor kit performance.
o  Always change pipet tips between liquid transfers. To avoid cross-contamination, we
recommend the use of aerosol-barrier pipet tips, o  All centrifugation steps are carried out at room temperature.
o  When working with chemicals, always wear a suitable lab coat, disposable gloves, and
protective goggles, o   Discard gloves if they become contaminated.
o   Do not combine components of different kits unless the lot numbers are identical, o  Avoid microbial contamination of the kit reagents.
o  To minimize the risk of infections from potentially infectious material, we recommend working
under laminar air-flow until the samples are lysed. o  This kit should only be used by trained personnel.
Preparing reagents and buffers
1. Adjust the thermomixer to 37°C.
2. Warm up the needed amount of Elution Buffer to 65°C (100 - 200 ul Elution Buffer are
needed per sample).
3. Heat heating blocks (e.g. thermomixer) to 56°C/ 65°C (depends on the protocol) and 95 °C.
4. Label the needed amount of 2.0 ml RTA Spin Filter Sets.
5. Label the needed amount of 1.5 ml Receiver Tubes (per sample: 1 Receiver Tube), add the needed amount of ethanol to the Wash Buffer I and II (see Kit Contents, page 3).
5 bacterial DNA extractions:
Binding Buffer B6, Wash Buffer I and II are ready to use! 50 bacterial DNA-extractions:
Add 21 ml 99.7% Isopropanol to the Binding Buffer B6. Mix by intensive shaking by inverting for 1 min. Shortly before use mix by inverting several times. Add 30 ml 96-100% ethanol to the bottle Wash Buffer I. Add 42 ml 96-100% ethanol to each bottle Wash Buffer II. Mix thoroughly and always keep the bottle firmly closed!
250 bacterial DNA-extractions:_
Add 84 ml 99.7% Isopropanol to the Binding Buffer B6. Mix by intensive shaking by inverting for 1 min. Shortly before use mix by inverting several times. Add 80 ml 96-100% ethanol to each bottle Wash Buffer I. Add 140 ml 96-100% ethanol to each bottle Wash Buffer II, Mix thoroughly and always keep the bottle firmly closed!
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RTP® Bacteria DNA Mini Kit 0419
Equipment and reagents to be supplied by user
When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDS). (See our webpage: www.invitek-molecular.com)
o Microcentrifuge
o Eppendorf Thermomixer (for 80°C)
o Measuring cylinder (250 ml)
o Disposable gloves
o Pipet with tips
o Reagents reservoirs for multichannel pipets
o 96-100 % ethanol
o 1.5 ml reaction tubes
o ddH20
o optional Octan
o optional centrifuge for 15 or 50 ml
o Isopropanol*
*The RTP® Bacteria DNA Mini Kit is validated with 2-Propanol; Rotipuran >99.7%, p.a., ACS, ISO (Order no. 6752) from Carl Roth
* Possible suppliers for Isopropanol:
Carl Roth Applichem
2-Propanol 2-Propanol für die Molekularbiologie
Rotipuran >99.7%, p.a., ACS, ISO Order no. A3928 Order no. 6752
Sigma
2-Propanol
Order no. 59304-1L-F
Important indication
Measurement of the DNA directly after isolation by intercalating fluorescent dyes may lead too low results. This is caused by the nature of the isolated DNA, due the denaturing step (95°C) during lysis DNA is partially single stranded. So fluorophores using double stranded DNA will not work correctly.
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RTP® Bacteria DNA Mini Kit 0419
Scheme of the RTP® Bacteria DNA Mini Kit
Please read the protocols carefully prior to the start of the preparation procedure!
transfer the sample into the Extraction Tube add 400 ul Resuspension Buffer
or
resuspend the sample in 400 ul Resuspension Buffer and transfer the complete mixture into the Extraction Tube incubate for 10 min at 37°C in a thermomixer (only gram-positive bacteria)
incubate for 10 min at 65°C in a thermomixer incubate for 5 -10 minutes at 95°C in a thermomixer
add 400 ul Binding Buffer B6 (follow preparing instructions) and mix the sample completely by pipetting up and down or by vortexing
transfer the sample on the RTA-Spin Filter incubate for 1 min
centrifuge for 2 min at 11.000 x g (11.000 rpm)
pipet 500 ul Wash Buffer I onto the RTA-Spin Filter centrifuge for 1 min at 11.000 x g (11.000 rpm) discard the flow-through and the RTA Receiver Tube
put the RTA Spin Filter in a new RTA Receiver Tube pipet 600 ul Wash Buffer II onto the RTA-Spin Filter centrifuge for 1 min at 11.000 x g (11.000 rpm) discard the flow-through and reuse the RTA Receiver Tube
to eliminate any traces of ethanol, centrifuge again for 4 min at maximum speed, discard the RTA Receiver Tube
transfer the RTA-Spin Filter into the 1.5 ml Elution Tube
pipet 200 ul of Elution Buffer (preheated to 65°C) directly onto the
center of the membrane of the RTA-Spin Filter
incubate for 1 min at RT
centrifuge for 1 min at 11.000 x g (11.000 rpm)
discard the RTA-Spin Filter
place the eluted bacterial DNA immediately in a refrigerator or store it at -20°C
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RTP® Bacteria DNA Mini Kit 0419
Instructions
The following notes are valid for all protocols:
Note: The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf.
The indicated rpm amounts are referring to this centrifuge.
Protocol 1:   Isolation of bacterial DNA from swab
Please read protocols prior the start of the preparation and complete preparing steps!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note: Switch on heating blocks (e.g. thermomixer) to 65°C and 95 °C 1a. For positive bacteria
Place the swab into the Extraction Tube and add 400 pi Resuspension Buffer R and mix by
stirring with the swab. Break or cut the swab and close the tube.
Incubate the sample in a thermomixer for 10 min at 37°C and at 65°C for 10 min (continuously shaking increases the lysis procedure, after 8 min its possible to switch the heating block to 65°C and do the heating of the block to 65° with the sample inside, if the block is not too slow > 4°C per min, you just do then incubation for 12 min, if your block is slower, you have to elongate incubation time).
Remove the swab. - Continue with step two. 1b. For gram negative bacteria
Place the swab into the Extraction Tube and add 400 pi Resuspension Buffer R and mix by
stirring with the swab. Break or cut the swab and close the tube.
Incubate the sample in a thermomixer for 10 min at 65°C for (continuously shaking increases the lysis procedure).
Remove the swab. - Continue with step two.
Note:  To get maximum yield of bacterial nucleic acids it is essential to leave the swab during the
complete lysis time into the reaction tube. It is possible to cut the shaft of the swab, so that you can close the cap of the Extraction Tube. It is also possible to do the lysis step with opened cap. The removing of the swab from the Extraction Tube ahead of time will be lead to a dramatically reduced final yield I
After lysis time carefully squeeze out the swab on the wall of the tube and discard the swab.
2. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min
(continuous shaking increases the lysis efficiency).
3. Add 400 pi Binding Buffer B6 to the sample and vortex shortly.
4. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
5. Add 500 pi Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube.
6. Add 600 pi Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
7. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 100 - 200 pi of Elution Buffer D. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11.000 x g.
Note:     The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected yield of bacterial DNA).
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RTP® Bacteria DNA Mini Kit 0419
Protocol 2: Isolation of DNA from bacteria pellets (1 x 109 bacteria cells)
Please read protocols prior the start of the preparation and complete preparing steps!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note:   Switch on heating blocks (e.g. thermomixer) to 65°C and 95 °C
Take an aliquot of the bacteria culture and spin it down at 11.000 x g (11.000 rpm) for 3 min. Remove the complete supernatant careful.
1a. For gram positive bacteria
Add 400 ul Resuspension Buffer R to the pellet and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer for 10 min at 37°C and at 65°C for 10 min (continuous shaking increases the lysis procedure, after 8 min its possible to switch the heating block to 65°C and do the heating of the block to 65° with the sample inside, if the block is not too slow > 4°C per min, you just do then incubation for 12 min, if your block is slower, you have to elongate incubation time).
Continue with step two.
1b. For gram negative bacteria
Add 400 ul Resuspension Buffer R to the pellet and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer at 65°C for 10 min (continuous shaking increases the lysis procedure). Continue with step two.
2. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency).
3. Add 400 ul Binding Buffer B6 to the sample and vortex shortly.
4. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
5. Add 500 ul Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube.
6. Add 600 ul Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
7. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 200 ul of Elution Buffer. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11.000 x g (11.000 rpm).
Note:      The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected yield of bacterial DNA).
14
RTP® Bacteria DNA Mini Kit 0419
Protocol 3:    Isolation of bacterial DNA e.g. Listeria ssp. from food samples
Please read protocols prior the start of the preparation and complete preparing steps!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note:    Switch on heating blocks (e.g. Thermomixer) to 65°C and 95 °C
How to Handle the food sample: § 35 LMBG
Take 25 g of the food material and homogenize the material. Add to the homogenized material 225 ml of the recommended culture media (e.g. Fraser media) and cultivate the recommended time (24h). Take a 1 ml aliquot of this culture and spin it down at 11.000 x g (11.000 rpm) for 3 min. Remove the complete supernatant careful.
1a. Gram positive bacteria
Add 400 ul Resuspension Buffer R to the pellet and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer for 10 min at 37°C and at 65°C for 10 min (continuous shaking increases the lysis procedure, after 8 min its possible to switch the heating block to 65°C and do the heating of the block to 65° with the sample inside, if the block is not too slow > 4°C per min, incubate for 12 min, if your block is slower, you have to elongate incubation time). Continue with step two.
1b. Gram negative bacteria
Add 400 ul Resuspension Buffer R to the pellet and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer at 65°C for 10 min (continuous shaking increases the lysis procedure). Continue with step two.
2. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency).
3. Add 400 ul Binding Buffer B6 to the sample and vortex shortly.
4. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
5. Add 500 ul Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube.
6. Add 600 ul Wash Buffer II and centrifuge at11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
7. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 200 ul of Elution Buffer. Incubate for 1 min at room temperature. Centrifuge for 1 min 11.000 x g (11.000 rpm).
Note: The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected
yield of bacterial DNA).
15
RTP® Bacteria DNA Mini Kit 0419
Protocol 4:   Isolation of DNA from periodonthopathogenic bacteria from paper points
Please read protocols prior the start of the preparation and complete preparing steps!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note: Switch on heating blocks (e.g. thermomixer) to 65°C and 95 °C
1. Transfer the paper point into the Extraction Tube.
Add 400 ul Resuspension Buffer R to the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer for 10 min at 37°C and at 65°C for 10 min (continuous shaking increases the lysis procedure, after 8 min its possible to switch the heating block to 65°C and do the heating of the block to 65° with the sample inside, if the block is not too slow > 4°C per min, you just do then incubation for 12 min, if your block is slower, you have to elongate incubation time).
2. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency).
After lysis centrifuge the sample at max. speed for 1 minute to spin down the paper point. Transfer the supernatant completely into a 1.5 ml reaction tube (not provided).
3. Add 400 ul Binding Buffer B6 to the sample and vortex shortly.
4. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
5. Add 500 ul Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube
6. Add 600 ul Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
7. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 200 ul of Elution Buffer (when one or two paper points were used) or add 400 ul Elution Buffer (when three or four paper points were used). Incubate for 1 min at room temperature.
Centrifuge for 1 min at 11.000 x g (11.000 rpm).
Note:     The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected yield of bacterial DNA).
Optional: If a white pellet is visible in the eluate, spin the sample again at maximum speed and transfer the supernatant into a clean tube !
16
RTP® Bacteria DNA Mini Kit 0419
Protocol 5:   Isolation of bacterial DNA from tissue biopsies
Please read protocols prior the start of the preparation and complete preparing steps!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note: Switch on heating blocks (e.g. thermomixer) to 56°C and 95 °C
1. Transfer 1 mg up to - max. 10 mg of the biopsy into the Extraction Tube. Add 400 ul of Resuspension Buffer R. Close the cap and vortex shortly.
Place the Extraction Tube into a thermomixer and incubate under continuously shaking for 30 - 60 min minutes at 56°C. Lysis time can be increased if the lysis is not completely.
2. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency).
After lysis centrifuge the sample at max. speed for 1 minute to spin down unlysed material. Transfer the supernatant completely into a 1.5 ml reaction tube (not provided).
3. Add 400 ul Binding Buffer B6 to the sample and vortex shortly.
4. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
5. Add 500 ul Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube
6. Add 600 ul Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate. Place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
7. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 80 - 120 ul of Elution Buffer (depends on expected yield and wished concentration of DNA). Incubate for 1 min at room temperature. Centrifuge for 1 min at 11.000 x g (11.000 rpm).
Note:      The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected yield of bacterial DNA).
17
RTP® Bacteria DNA Mini Kit 0419
Protocol 6:  Isolation of bacterial DNA from paraffin embedded tissue
Please read protocols prior the start of the preparation and complete preparing steps!!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note: Switch on heating blocks (e.g. thermomixer) to 56°C and 95 °C
1. Transfer the sample into a 1.5 ml reaction tube. Add 1 ml Octane and vortex carefully to dissolve the paraffin. Follow the dissolution until the tissue sample looks transparent (while paraffin is still white). Centrifugation for 2 min at maximum speed to collect the tissue sample. Discard the supernatant very careful. This step should be repeated if any paraffin is still remains in the sample. Add 0.5 ml 96 -100 % Ethanol to the tissue sample and mix the tube thoroughly. Short centrifugation and removing of the ethanol by aspiration with a pipette. Then incubate the open tube at 56°C to evaporate the residual ethanol.
2. Transfer the deparaffinized tissue sample into the Extraction Tube. Add 400 ul of Resuspension Buffer R. Close the cap and vortex shortly. Place the Extraction Tube
into a     thermomixer and incubate under continuously shaking for 30 - 60 min at 56°C. Lysis time can be increased if the lysis is not completely.
3. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency). After lysis centrifuge the sample at max. speed for 1 minute to spin down unlysed material. Transfer the supernatant completely into a 1.5 ml reaction tube.
4. Add 400 ul Binding Buffer B6 to the sample and vortex shortly.
5. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
6. Add 500 ul Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube. Place the RTA Spin Filter into a new RTA Receiver Tube.
7. Add 600 ul Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
8. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 80 - 120 ul of Elution Buffer D (depends on expected yield and wished concentration of DNA). Incubate for 1 min at room temperature. Centrifuge for 1 min at 11.000 x g (11.000 rpm).
Note:   The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected yield of bacterial DNA).
18
RTP® Bacteria DNA Mini Kit 0419
Protocol 7:   Isolation of bacterial DNA from urine sample
Please read protocols prior the start of the preparation and complete preparing steps!
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note:    Switch on heating blocks (e.g. thermomixer) to 65°C and 95 °C
1. Centrifugation of the collected urine sample (15 - 50 ml) for 15 minutes at 3500 rpm.
Decant the supernatant carefully, resuspend the sediment with 3 ml 1 x PBS and centrifuge for5 minutes at 3.500 rpm. Decant the supernatant completely by carefully inverting the tube for some minutes.
It is important to remove the supernatant completely! Residual amounts of liquid will have negative influence on the further extraction procedure!
2a. For gram positive bacteria
Add 400 ul Resuspension Buffer R to the sediment and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer for 10 min at 37°C and at 65°C for 10 min (continuous shaking increases the lysis procedure, after 8 min its possible to switch the heating block to 65°C and do the heating of the block to 65° with the sample inside, if the block is not too slow > 4°C per min, you just do then incubation for 12 min, if your block is slower, you have to elongate incubation time). Continue with step 3.
2b. For gram negative bacteria
Add 400 ul Resuspension Buffer R to the sediment and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer at 65°C for 10 min (continuous shaking increases the lysis procedure). Continue with step 3.
3. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency).
4. Add 400 ul Binding Buffer B6 to the sample and vortex shortly.
5. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
6. Add 500 ul Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube.
7. Add 600 ul Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
8. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 100 ul of Elution Buffer. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11.000 x g (11.000 rpm).
Note: The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected
yield of bacterial DNA).
19
RTP® Bacteria DNA Mini Kit 0419
Protocol 8:   Isolation of bacterial DNA from water sample (big volume)
Please read protocols prior the start of the preparation and complete preparing steps!_
Attention: Please be aware, that you have to prepare the Binding Buffer B6 - see instruction page: 10 Important note: Switch on heating blocks (e.g. thermomixer) to 65°C and 95 °C
1. Concentrate the starting sample (e.g. 1 liter) by standard procedures like centrifugation or filtration. Finally spin down the bacteria by centrifugation e.g. using a 50 ml reaction tube. Decant the supernatant carefully and resuspend the sediment with 10 ml 1 x PBS. Centrifugation for 5 minutes at 3500 rpm. Decant the supernatant carefully but completely by inverting the tube for some minutes.
It is important to remove the supernatant completely! Residual amounts of liquid will have negative influence on the further extraction procedure!
2a. For gram positive bacteria
Add 400 pi Resuspension Buffer R to the sediment and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer for 10 min at 37°C and at 65°C for 10 min (continuous shaking increases the lysis procedure, after 8 min its possible to switch the heating block to 65°C and do the heating of the block to 65° with the sample inside, if the block is not too slow > 4°C per min, you just do then incubation for 12 min, if your block is slower, you have to elongate incubation time). Continue with step 3.
2b. For gram negative bacteria
Add 400 pi Resuspension Buffer R to the sediment and resuspend the pellet by pipetting up and down. Transfer the resuspended sample into the Extraction Tube and vortex shortly. Incubate the sample in a thermomixer at 65°C for 10 min (continuous shaking increases the lysis procedure). Continue with step 3.
3. Place the Extraction Tube into a thermomixer and incubate at 95°C for 5 -10 min (continuous shaking increases the lysis efficiency).
4. Add 400 pi Binding Buffer B6 to the sample and vortex shortly.
5. Load the sample onto the RTA Spin Filter Set and incubate for 1 min. Centrifuge at 11.000 x g (11.000 rpm) in a standard table centrifuge for 2 min. Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube.
6. Add 500 pi Wash Buffer I and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate, and the RTA Receiver Tube.
Place the RTA Spin Filter into a new RTA Receiver Tube
7. Add 600 pi Wash Buffer II and centrifuge at 11.000 x g (11.000 rpm) for 1 min. Discard the filtrate. Place the RTA Spin Filter back into the RTA Spin Filter and finally centrifuge for 4 min at max. speed to remove the ethanol completely.
8. Place the RTA Spin Filter into a new 1.5 ml Receiver Tube and add 100 pi of Elution Buffer. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11.000 x g (11.000 rpm).
Note:       The DNA can also be eluted with a lower volume of Elution Buffer (depends on the expected yield of bacterial DNA).
ATTENTION: A specialized kit for Legionella ssp. is available on request!
20
RTP® Bacteria DNA Mini Kit 0419
Troubleshooting
Problem	Probable cause	Comments and suggestions
clogged Spin-Filter	insufficient lysis and/or too much starting material	increase lysis time increase centrifugation speed reduce amount of starting material
low amount of extracted DNA	insufficient lysis incomplete elution insufficient mixing of the sample with Binding Solution	increase lysis time reduce amount of starting material overloading of Spin Filter reduces yield prolong the incubation time with Elution Buffer to 5 -10 min do the elution steps twice take higher volume of Elution Buffer mix sample sufficient by pipetting up and down or vortexing with Binding Buffer B6 prior to transfer the sample onto the Spin Filter
low concentration of extracted DNA	too much Elution Buffer incorrect storage of starting material	elute the DNA twice with lower volume of Elution Buffer ensure that the storage of starting material was correctly avoid thawing of the material
degraded DNA	incorrect storage of starting material old material	ensure that the starting material is fresh or stored under appropriate condition (for long time storage at - 20°C) avoid thawing and freezing of the material old material often contains degraded DNA
DNA does not perform well in downstream-applications (e.g. RT-PCR or PCR)	ethanol carryover during elution salt carryover during elution	increase time for removing of ethanol ensure that the Wash Buffers are at room temperature check up the Wash Buffers for salt precipitates if there are any precipitates, solve these precipitates by careful warming
21
RTP® Bacteria DNA Mini Kit 0419
Appendix
General notes on handling DNA Nature of DNA
The length and delicate physical nature of DNA require careful handling to avoid damage due to shearing and enzymatic degradation. Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments, high temperature, and UV irradiation. Careful isolation and handling of high molecular weight DNA is necessary to ensure it will function well in various downstream applications. Damaged DNA could perform poorly in applications such as genomic Southern blotting, long-template PCR, and construction of cosmid libraries.
Handling    fresh    and    stored    material    before    the    extraction    of DNA
For the isolation of genomic DNA from cells or tissues, use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at -70°C. This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases.
Storage of DNA
Store genomic DNA at +2 to +8°C. Storing genomic DNA at -15 to -25°C can cause shearing of DNA, particularly if the DNA is exposed to repeated freeze-thaw cycles. Plasmid DNA and other small circular DNA's can be stored at +2 to +8°C or at -15 to -25°C.
Handling of DNA
Avoid vigorous pipetting. Pipetting genomic DNA through small tip openings causes shearing or nicking. One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA.
DNA Yield
The amount of purified bacterial DNA depends on the microorganism content, sample source, transport, storage and age. Various different primary tubes and anticoagulants (except heparin) can be used to collect samples for the Invisorb® procedure.
22
RTP® Bacteria DNA Mini Kit 0419
Ordering information
Product Package size
RTP® Bacteria DNA Mini Kit RTP® Bacteria DNA Mini Kit RTP® Bacteria DNA Mini Kit
5 extractions 50 extractions 250 extractions
Single components
Extraction Tube 10 tubes Binding Buffer B6 (add 21 ml)     9 ml
Resuspension Buffer R 30 ml
Wash Buffer I 30ml
Wash Buffer II 18 ml
Elution Buffer 15 ml
Related Products
InviMag® Bacteria DNA Mini Kit/ KFmL InviMag® Bacteria DNA Mini Kit/ KFmL
Catalogue No.
1033200100 1033200200 1033200300
1033201500 1033202000 1033202200 1033203500 1033203600 1033204000
75 purifications 300 purifications
2433150200 2433150400
Possible suppliers for Isopropanol:
Carl Roth Applichem Sigma
2-Propanol 2-Propanol für die Molekularbiologie 2-Propanol
Rotipuran >99.7%, p.a., ACS, ISO 0rder no A3928 Order no. 59304-1L-F Order no. 6752
Further applications:
1. Isolation of DNA from Mycobacteria from sputum, bronchial lavage, tissue samples or BALB samples.
Please use the RTP® Mycobacteria Kit
2. Isolation of bacterial DNA from stool sample
Please use the PSP® Spin Stool DNA or the PSP® Spin Stool DNA PLUS Kit
3. Automatic isolation of bacterial DNA
Please contact our technical support.
4. Isolation of DNA from Legionella species
Please contact our technical support.
23
RTP® Bacteria DNA Mini Kit 0419
INVITEK
Molecular
Invitek Molecular GmbH Robert-Rbssle-Str. 10 13125 Berlin, Germany
Phone: +49 30 94 89 29 01
Fax:+49 30 94 89 29 09
E-mail: info@invitek-molecular.com
www.invitek-molecular.com §
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