Manual innuPREP DNA Mini Kit

Instructions for Use
Life Science Kits & Assays
innuPREP DNA Mini Kit

Order No.:
845-KS-1041010 reactions
845-KS-1041050 reactions
8.61041250 reactions
Publication No.: HB_KS-_e_190823
This documentation describes the state at the time of publishing.

It needs not necessarily agree with future versions. Subject to change!
Print-out and further use permitted with indication of source.
© Copyright 2019, Analytik Jena AG, AJ Innuscreen GmbH
Manufacturer:
AJ Innuscreen GmbH
Robert-Rössle-Straße 10
13125 Berlin · Germany
Made in Germany!
Distribution/Publisher: Phone +49 3641 77 9400

Analytik Jena AG Fax +49 3641 77 767776
Konrad-Zuse-Straße 1 www.analytik-jena.com
07745 Jena · Germany [email protected]
Introduction
Contents
1 Introduction ...................................................................................................... 2
1.1 Intended use ........................................................................................... 2
1.2 Notes on the use of this manual........................................................... 2
2 Safety precautions............................................................................................ 3
3 Storage conditions ........................................................................................... 4
4 Functional testing and technical assistance .................................................. 5
5 Product use and warranty ............................................................................... 5
6 Kit components ................................................................................................ 6
6.1 Included kit components ....................................................................... 6
6.2 Components not included in the kit ..................................................... 7
7 Product specifications ...................................................................................... 7
8 Initial steps before starting ............................................................................. 8
9 Protocols for Isolation of genomic DNA ......................................................... 8
9.1 Protocol 1: Isolation from tissue samples or rodent tails ................... 8
9.2 Protocol 2: Isolation from paraffin embedded tissue samples ........10
9.3 Protocol 3: Isolation from buccal swab ..............................................12
9.4 Protocol 4: Isolation from cell cultures ..............................................14
10 Troubleshooting ............................................................................................. 16
innuPREP DNA Mini Kit Issue 08 / 2019

1
Introduction
1 Introduction
1.1 Intended use
The innuPREP DNA Mini Kit has been designed as a very efficient tool for
fast isolation of genomic DNA from different amounts and different types
of starting materials like tissue samples up to 50 mg, paraffin-embedded
tissue material, buccal swabs, mouse or rodent tail, eukaryotic cell pellets.
The kit is intended for use by professional users. The extraction procedure
is based on a new kind of chemistry, which combines an extremely fast
lysis step with a subsequent efficient binding of genomic DNA on a Spin
Filter surface following washing of the bound DNA and finally eluting of
the DNA. The recovery of DNA and the quality are excellent. Extracted
DNA is available approx. 15 minutes after lysis of starting material. The
isolated DNA is suitable for a wide range of different downstream appli-
cations like amplification reactions and further analytical procedures. Di-
agnostic results generated using the extraction procedure in conjunction
with diagnostic tests should be interpreted with regard to other clinical or
laboratory results. To reduce irregularities in diagnostic results, internal
controls for downstream applications should be used.
CONSULT INSTRUCTION FOR USE

This package insert must be read carefully prior to use. Package insert
instructions must be followed accordingly. Reliability of results cannot
be guaranteed if there are any deviations from the instructions in this
package insert.
1.2 Notes on the use of this manual
For easy reference and orientation, the manual uses the following warn-
ing and information symbols as well as the shown methodology:
Symbol Information
REF
Catalogue number.
Content
N Contains sufficient reagents for <N> reactions.

2
Safety precautions
Symbol Information
Storage conditions
Store at room temperature or shown conditions respectively.
Consult instructions for use

This information must be observed to avoid improper use of the kit
and the kit components.
Expiry date
Lot number
The number of the kit charge.
Manufactured by
Contact information of manufacturer.
For single use only

Do not use components for a second time.
The following systematic approach is introduced in the manual:

 The chapters and figures are numbered consecutively.
 A cross reference is indicated with an arrow (e.g.  „Notes on the
use of this manual“ p. 2).
 Working steps are numbered.
2 Safety precautions
NOTE
Read through this chapter carefully prior to guarantee your own safety
and a trouble-free operation.
Follow all the safety instructions explained in the manual, as well as all
messages and information, which are shown.
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.

3
Storage conditions
FOR SINGLE USE ONLY!

This kit is made for single use only!
ATTENTION!
Don’t eat or drink components of the kit!
The kit shall only be handled by educated personnel in a laboratory envi-
ronment!
If the buffer bottles are damaged or leaking, wear gloves and protective
goggles when discarding the bottles in order to avoid any injuries. This kit
could be used with potential infectious samples. Therefore, all liquid
waste must be considered as potentially infectious and must be handled
and discarded according to local safety regulation.
Please observe the federal, state and local safety and environmental
regulations. Follow the usual precautions for applications using extracted
nucleic acids. All materials and reagents used for DNA or RNA isolation
should be free of DNases or RNases.
ATTENTION!
Do not add bleach or acidic components to the waste after sample prepa-
ration!
NOTE
Emergency medical information in English and German can be obtained
24 hours a day from:
Poison Information Center, Freiburg / Germany
Phone: +49 (0)761 19 240.
For more information on GHS classification please download the Safety

Data Sheet (SDS) from our website (www.analytik-jena.com). Find the
SDS of each product and its components in the "downloads"-section.
3 Storage conditions
Store lyophilized Proteinase K at 4 °C to 8 °C! Divide dissolved

Proteinase K into aliquots and store at -22 °C to -18 °C. Repeated freezing
and thawing will reduce the activity dramatically!
4
Functional testing and technical assistance
All other components of the innuPREP DNA Mini Kit should be stored dry
at room temperature (15 °C to 30 °C). When stored at room temperature,
the kit is stable until the expiration date printed on the label on the kit
box.
Before every use make sure that all components have room temperature.
If there are any precipitates within the provided solutions dissolve these
precipitates by careful warming.
4 Functional testing and technical assistance
The Analytik Jena AG guarantees the correct function of the kit for appli-
cations as described in the manual. This product has been produced and
tested in an ISO 13485 certified facility.
We reserve the right to change or modify our products to enhance their
performance and design. If you have any questions or problems regarding
any aspects of the innuPREP DNA Mini Kit or other Analytik Jena AG
products, please do not hesitate to contact us. For technical support or
further information in Germany please dial +49 36 41 / 77 94 00. For
other countries please contact your local distributor.
5 Product use and warranty
The kit is not designed for the usage of other starting materials or other
amounts of starting materials than those, referred to in the manual
( “Product specifications“ p. 7). Since the performance characteristics of
Analytik Jena AG kits have just been validated for the application de-
scribed above, the user is responsible for the validation of the perfor-
mance of Analytik Jena AG kits using other protocols than those described
below. Analytik Jena AG kits may be used in clinical diagnostic laboratory
systems after the laboratory has validated the complete diagnostic system
as required by CLIA’ 88 regulations in the U.S. or equivalents in other
countries.

5
Kit components
All products sold by Analytik Jena AG are subjected to extensive quality

control procedures and are warranted to perform as described when used
correctly. Any problems should be reported immediately.
NOTE
The kit is for research use only!
6 Kit components
6.1 Included kit components
10 50 250
845-KS-1041010 845-KS-1041050 845-KS-1041250
Lysis Solution TLS 5 ml 25 ml 120 ml
Binding Solution TBS 5 ml 25 ml 120 ml
Proteinase K for 1 x 0.3 ml for 1 x 1.5 ml for 5 x 1.5 ml

working solution working solution working solution
Washing Solution HS 3 ml 15 ml 70 ml
(conc.)
Washing Solution MS 3 ml 15 ml 60 ml
(conc.)
Elution Buffer 2 x 2 ml 15 ml 60 ml
Spin Filter 10 50 5 x 50
Receiver Tubes 40 4 x 50 20 x 50
Elution Tubes 10 50 5 x 50
Manual 1 1 1

6
Product specifications
6.2 Components not included in the kit
 Xylene or Octan (for paraffin embedded tissue samples)

 1.5 ml and 2.0 ml tubes
 ddH2O for dissolving Proteinase K
 96–99.8 % ethanol (molecular biology grade, undenaturated)
 RNase A (10 mg/ml); optional
7 Product specifications
Starting material:
 Tissue samples (up to 50 mg)
 Rodent tail (up to 1 cm)
 Paraffin embedded tissue samples
 Buccal swabs
 Eukaryotic cells (up to 5 x 106 cells)
Time for isolation:

 Approximately 8 minutes after lysis step
Typical yield:
 Depends on type and amount of starting material
 The extracted genomic DNA (gDNA) can be used for a wide range of
different molecular biology applications.
 Binding capacity of the spin column is > 100 µg gDNA

7
Initial steps before starting
8 Initial steps before starting
 Heat thermal mixer or water bath (paraffin embedded samples:

37 °C, followed by 50 °C and 90 °C; buccal swab/ cell cultures: 50 °C).
 Add to Proteinase K the indicated amount of ddH2O to each vial, mix
thoroughly and store as described above.
845-KS-1041010 Add 0.3 ml ddH2O to lyophilized Proteinase K.
 Add to Washing Solution HS (conc.) the indicated amount of abso-

lute ethanol. Mix thoroughly and store as described above.
845-KS-1041010 Add 3 ml ethanol to 3 ml Washing Solution HS (conc.).
 Add to Washing Solution MS (conc.) the indicated amount of abso-

lute ethanol. Mix thoroughly and store as described above.
845-KS-1041010 Add 7 ml ethanol to 3 ml Washing Solution MS (conc.).
845-KS-1041050 Add 35 ml ethanol to 15 ml Washing Solution MS (conc.).
 Centrifugation steps should be carried out at room temperature.

 Avoid freezing and thawing of starting material.
9 Protocols for Isolation of genomic DNA
9.1 Protocol 1: Isolation from tissue samples or rodent tails
1. Cut max. 50 mg of tissue sample or up to 1 cm of rodent tail into

small pieces and place the tissue in a 1.5 ml or 2.0 ml reaction tube.
Add 400 µl Lysis Solution TLS and 25 µl Proteinase K, mix vigorous-
ly by pulsed vortexing for 5 seconds Incubate at 50 °C until the sam-
ple is completely lysed (approx. 1–2 hours; especially for rodent tails
use not more than 2 hours for lysis).
8
Protocols for Isolation of genomic DNA
NOTE
We recommend to use a shaking platform (thermal mixer, water bath or
another rocking platform) for a continuous shaking of the sample. Alter-
natively, vortex the sample every 10 minutes during the incubation. No
shaking will reduce the lysis efficiency.
2. Centrifuge the 1.5 ml tube at 11,000 x g (~11,000 rpm) for

1 minute to spin down unlysed material. Transfer the supernatant
into another 1.5 ml tube.
NOTE
To remove RNA from the sample (if necessary) add 2 µl of RNase A solu-
tion (10 mg/ml), vortex shortly and incubate for 5 minute at RT.
3. Add 400 µl Binding Solution TBS to the lysed sample, mix by brief
vortexing for 15 seconds.
IMPORTANT
Please be careful! Mechanical stress by vortexing or extensive mixing
leads to shearing of high-molecular weight chromosomal DNA. But it is
important that the sample and the Binding Solution TBS are mixed com-
pletely to get a homogeneous solution.
4. Apply the sample to the Spin Filter located in a Receiver Tube. Close
the cap and centrifuge at 11,000 x g (~11,000 rpm) for 2 minutes.
Discard the Receiver Tube with the filtrate. Place the Spin Filter into
a new Receiver Tube.
NOTE
If the solution has not completely passed through the Spin Filter, centri-
fuge again at higher speed or prolong the centrifugation time.
5. Open the Spin Filter and add 500 µl Washing Solution HS, close the
cap and centrifuge at 11,000 x g (~11,000 rpm) for 1 minute. Dis-
card the Receiver Tube with the filtrate. Place the Spin Filter into a
new Receiver Tube.
6. Open the Spin Filter and add 750 µl Washing Solution MS, close the
9
new Receiver Tube.
7. Centrifuge at 11,000 x g (11,000 rpm) for 3 minutes to remove all
traces of ethanol. Discard the Receiver Tube.
8. Place the Spin Filter into an Elution Tube. Carefully open the cap of
the Spin Filter and add 200 µl Elution Buffer. Incubate at room tem-
perature for 1 minute. Centrifuge at 11,000 x g (~11,000 rpm) for
1 minute. A second elution step will increase the yield of extracted
DNA.
NOTE
The DNA can be eluted with a lower or a higher volume of Elution Buffer
(depends on the expected yield of genomic DNA). Elution with lower vol-
umes of Elution Buffer increases the final concentration of DNA. Store
the extracted DNA at 4 °C to 8 °C. For long time storage placing at –18 °C
to -22 °C is recommended.
9.2 Protocol 2: Isolation from paraffin embedded tissue samples
1. Place a piece of starting material into a 2.0 ml tube, add 1 ml Octane

or Xylene and vortex carefully to dissolve the paraffin. Follow the
dissolution until the tissue sample looks transparent (while paraffin
remains white).
2. Centrifuge at maximum speed for 5 minutes at room temperature.
Discard the supernatant very carefully by aspirating with a pipette.
IMPORTANT
Do not remove the pellet!
NOTE
This step should be repeated if any paraffin is still in the sample.
3. Add 1 ml ethanol (96–99.8 %) to the pellet and vortex vigorously.

4. Centrifuge at maximum speed at room temperature for 3 minutes
and remove the ethanol by pipetting.

10
IMPORTANT
Do not remove the pellet!
Repeat the washing step with ethanol once again.

Incubate the open tube at 37 °C for 10–15 minutes to evaporate the
residual ethanol completely.
Add 400 µl Lysis Solution TLS and 25 µl Proteinase K, mix vigorous-
ly by pulsed vortexing for 5 seconds Incubate at 50 °C until the sam-
ple is completely lysed.
Pre-heat the thermal mixer without the sample to 90 °C, afterwards
incubate the lysed sample for 60 minutes at 90 °C.
Add 400 µl Binding Solution TBS to the lysed sample, mix by brief
IMPORTANT
Please be careful! Mechanical stress by vortexing or extensive mixing
leads to shearing of high-molecular weight chromosomal DNA. But it is
important that the sample and the Binding Solution TBS are mixed com-
pletely to get a homogeneous solution.
Apply the sample to the Spin Filter located in a Receiver Tube. Close
NOTE
Discard the Receiver Tube with the filtrate. Place the Spin Filter into
Open the Spin Filter and add 500 µl Washing Solution HS, close the
new Receiver Tube.
Open the Spin Filter and add 750 µl Washing Solution MS, close the
new Receiver Tube.

11
Centrifuge at 11,000 x g (11,000 rpm) for 3 minutes to remove all

Place the Spin Filter into an Elution Tube. Carefully open the cap of
the Spin Filter and add 50–100 µl Elution Buffer. Incubate at room
temperature for 1 minute. Centrifuge at 11,000 x g (11,000 rpm)
for 1 minute. A second elution step will increase the yield of extract-
ed DNA.
NOTE
Store the extracted DNA at 4 °C to 8 °C, for long time storage at -18 °C to
-22 °C.
9.3 Protocol 3: Isolation from buccal swab
IMPORTANT
To get maximum yield of DNA it is essential to leave the swab during the
complete lysis time in the 1.5 ml tube. It is possible to cut the shaft of
the swab, so that you can close the cap of the tube. The removal of the
swab from the tube ahead of time will lead to a dramatically reduced fi-
nal yield!
1. Place the swab into a 1.5 ml reaction tube. Add 400 µl Lysis Solution
TLS and 25 µl Proteinase K, mix vigorously by pulsed vortexing for
5 seconds. Incubate at 50 °C for 10–15 minutes.
NOTE
natively, vortex the sample 3-4 times during the incubation. No shaking
will reduce the lysis efficiency.
2. After lysis time remove the swab from the tube and squeeze the
swab on the wall of the tube to remove all Lysis Solution TLS from
the swab.

12
IMPORTANT
Mechanical stress by vortexing or extensive mixing leads to shearing of
high-molecular weight chromosomal DNA. But it is important that the
sample and the Binding Solution TBS are mixed completely to get a ho-
mogeneous solution.
NOTE
5. Discard the Receiver Tube with the filtrate. Place the Spin Filter into
new Receiver Tube.
new Receiver Tube.
DNA.
NOTE
the extracted DNA at 4 °C to 8 °C. For long time storage placing at -18 °C
13
9.4 Protocol 4: Isolation from cell cultures
1. Pellet up to 5 x 106 cells by centrifugation for 10 minutes at

5,000 x g (~5,000 rpm). Discard supernatant. Add 400 µl Lysis Solu-
tion TLS and 25 µl Proteinase K to the pellet, mix vigorously by
pulsed vortexing for 5 seconds. Incubate at 50 °C until the sample is
completely lysed.
NOTE
natively, vortex the sample 3-4 times during the incubation. No shaking
will reduce the lysis efficiency.
IMPORTANT
Mechanical stress by vortexing or extensive mixing leads to shearing of
high-molecular weight chromosomal DNA. But it is important that the
sample and the Binding Solution TBS are mixed completely to get a ho-
mogeneous solution.
NOTE
4. Discard the Receiver Tube with the filtrate. Place the Spin Filter into
new Receiver Tube.
14
new Receiver Tube.
DNA.
NOTE
the extracted DNA at 4 °C to 8 °C. For long time storage placing at -18 °C

15
Troubleshooting
10 Troubleshooting
Problem / probable cause Comments and suggestions

Clogged Spin Filter
Insufficient lysis and/or too much Increase lysis time.
starting material Increase centrifugation speed.
After lysis centrifuge the lysate to pellet un-
lysed material.
Reduce amount of starting material.
Low amount of extracted DNA
Insufficient lysis Increase lysis time!
Reduce amount of starting material. Over-
loading reduces yield!
Incomplete elution Prolong the incubation time with Elution
Buffer to 5 minutes or repeat elution step
once again.
Take a higher volume of Elution Buffer.
Insufficient mixing with Binding So- Mix sample with Binding Solution TBS by
lution TBS pipetting or by vortexing prior to transfer of
the sample onto the Spin Filter.
Low concentration of extracted DNA
Too much Elution Buffer was used in Elute the DNA with lower volume of Elution
the elution step Buffer
Degraded or sheared DNA
Incorrect storage of starting mate- Ensure that the starting material is frozen
rial immediately after taking in liquid nitrogen
or at -18 °C to -22° C! For long time storage
continuously store at -78 °C to -82° C! Avoid
thawing of the material.
Old starting material Old material often contains degraded DNA.
Repeat with fresh material.
RNA contamination
Extracted DNA is contaminated with Perform an RNase A digestion.
RNA

16
engl. 0/1 – Analytik Jena AG, Jena
Headquarters
© Analytik Jena AG
Analytik Jena AG Pictures: Analytik Jena AG

Konrad-Zuse-Str. 1 Subjects to changes in design and scope of delivery as well as further technical development!
07745 Jena · Germany
Phone +49 3641 77 70

Fax +49 3641 77 9279
[email protected]
www.analytik-jena.com

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Instructions for Use

Life Science Kits & Assays

innuPREP DNA Mini Kit

Publication No.: HB_KS-_e_190823

This documentation describes the state at the time of publishing.

Distribution/Publisher: Phone +49 3641 77 9400

innuPREP DNA Mini Kit Issue 08 / 2019

1.1 Intended use

CONSULT INSTRUCTION FOR USE

1.2 Notes on the use of this manual

innuPREP DNA Mini Kit Issue 08 / 2019

Consult instructions for use

For single use only

The following systematic approach is introduced in the manual:

innuPREP DNA Mini Kit Issue 08 / 2019

FOR SINGLE USE ONLY!

For more information on GHS classification please download the Safety

Store lyophilized Proteinase K at 4 °C to 8 °C! Divide dissolved

4 Functional testing and technical assistance

5 Product use and warranty

innuPREP DNA Mini Kit Issue 08 / 2019

All products sold by Analytik Jena AG are subjected to extensive quality

6.1 Included kit components

Lysis Solution TLS 5 ml 25 ml 120 ml

Binding Solution TBS 5 ml 25 ml 120 ml

Proteinase K for 1 x 0.3 ml for 1 x 1.5 ml for 5 x 1.5 ml

innuPREP DNA Mini Kit Issue 08 / 2019

6.2 Components not included in the kit

 Xylene or Octan (for paraffin embedded tissue samples)

Time for isolation:

innuPREP DNA Mini Kit Issue 08 / 2019

8 Initial steps before starting

 Heat thermal mixer or water bath (paraffin embedded samples:

 Add to Washing Solution HS (conc.) the indicated amount of abso-

 Add to Washing Solution MS (conc.) the indicated amount of abso-

 Centrifugation steps should be carried out at room temperature.

9 Protocols for Isolation of genomic DNA

9.1 Protocol 1: Isolation from tissue samples or rodent tails

1. Cut max. 50 mg of tissue sample or up to 1 cm of rodent tail into

2. Centrifuge the 1.5 ml tube at 11,000 x g (~11,000 rpm) for

9.2 Protocol 2: Isolation from paraffin embedded tissue samples

1. Place a piece of starting material into a 2.0 ml tube, add 1 ml Octane

3. Add 1 ml ethanol (96–99.8 %) to the pellet and vortex vigorously.

innuPREP DNA Mini Kit Issue 08 / 2019

Repeat the washing step with ethanol once again.

innuPREP DNA Mini Kit Issue 08 / 2019

Centrifuge at 11,000 x g (11,000 rpm) for 3 minutes to remove all

9.3 Protocol 3: Isolation from buccal swab

innuPREP DNA Mini Kit Issue 08 / 2019

9.4 Protocol 4: Isolation from cell cultures

1. Pellet up to 5 x 106 cells by centrifugation for 10 minutes at

innuPREP DNA Mini Kit Issue 08 / 2019

Problem / probable cause Comments and suggestions

innuPREP DNA Mini Kit Issue 08 / 2019

Analytik Jena AG Pictures: Analytik Jena AG

Phone +49 3641 77 70

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Publication No.: HB_KS-_e_190823