User manual / Technical Information

USER MANUAL
Roti®-Prep Blood Genomic
DNA MINI
For isolation of DNA from whole blood
Ord. No. 8620
Roti®-Prep Blood Genomic DNA MINI
Carl Roth GmbH + Co. KG
Roti®-Prep Blood Genomic DNA MINI
For isolation of DNA from whole blood
Introduction and product description:
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Preparation by well-known mini spin-column system
Fast, easy and efficient
Preparation in approx. 25 minutes
Yield up to 30 µg gDNA
Roti®-Prep Blood Genomic DNA MINI has been specially designed for the isolation of high
purity genomic DNA from blood samples.
The isolation is carried out through the proven and reliable spin column technique, which is
very easy to handle and needs only few steps.
The Roti®-Prep Blood Genomic DNA MINI kit has been adapted to gain high purity DNA from
fresh or frozen blood (EDTA or citrate stabilised) or whole blood, respectively. In order to get
optimal results, two protocols, depending on sample amount, are provided. Furthermore,
the properties of the binding buffer have been optimised to reach highest binding capacity
with blood samples, permitting a yield of up to 30 µg gDNA out of 400 µl blood.
Utilisation is uncomplicated and fast to achieve (approx. 25 minutes, lysis included), and
offers best conditions for following downstream applications.
Isolated gDNA is free of EDTA, RNA, or proteins, and may directly be applied to all standard
downstream applications like, for instance, restriction digest, PCR etc.
For research use only.
The kit is intended for use by professional users and does not provide a diagnostic result. It
is the sole responsibility of the user to use and validate the kit in conjunction with all
required downstream in vitro assays.
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.
Roti®-Prep Blood Genomic DNA MINI
Carl Roth GmbH + Co. KG
Cautions:
Lysis Buffer LSN*
Warning H319-H400
Binding Buffer BR
Danger H225-H319-H336
P210-P280-P303+P361+P353-P305+P351+P338-P312
Proteinase K
P273-P280-P305+P351+P338
Danger H315-H319-H334-H317-H335
P280-P284-P333+P313-P337+P313-P342+P311
Washing Solution WSO
Danger H225-H319-H336
P210-P280-P303+P361+P353-P305+P351+P338-P312
*In case of forming of precipitates, please dissolve by careful warming.
MSDS: the appropriate MSDS can be downloaded from our website www.carlroth.com.
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.
Literature Citation: When describing a procedure for publication using this product, please
refer to it as Carl Roth’s Roti®-Prep Blood Genomic DNA MINI Kit.
1. Materials provided with the kit and storage conditions
Amount
6 / 2x30 / 6x30 mg
12 / 25 / 120 ml
8 / 50 / 250 ml
8 / 30 / 120 ml
2 / 10 / 2x18 ml
2x2 / 15 / 2x30 ml
10 / 50 / 250
50/ 250 / 1.250
10 / 50 / 250
Component
Proteinase K
Lysis Buffer LSN
Binding Buffer BR
Washing Solution WSO (ready-to-use)
Washing Buffer WSH (Base)
Elution Buffer EB
Mini spin columns (red)
1.5 ml Elution tubes
2 ml Collection tubes
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Storage
+4 °C / -20 °C*
>20 °C**
RT
RT
RT
RT
RT
RT
RT
Carl Roth GmbH + Co. KG
Roti®-Prep Blood Genomic DNA MINI
* Lyophilized Proteinase K should be stored at +4 °C. Prior to use, dissolve Proteinase K in 0.3
/ 1.5 / 5x1.5 ml sterile, distilled water as given below. Dissolved Proteinase K should be
stored in aliquots at -20 °C. Avoid repeated freeze&thaw cycles for each tube.
** Lysis Buffer should be stored at over 20 °C in order to prevent precipitation of reagents.
The Roti®-Prep Blood Genomic DNA MINI Kit should be stored dry, at room temperature (1425 °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 components of each Roti®-Prep Blood Genomic DNA MINI Kit were tested by isolation of
genomic DNA from whole blood samples in general, followed by spectro-photometrical
measurement, gel electrophoresis and target-amplification. The user is responsible,
however, to validate the performance of the Roti®-Prep Blood Genomic DNA MINI Kit for any
particular use, since the performance characteristics of our kits have not been validated for
specific applications.
2. Required Material and Equipment not included in this kit
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Ethanol 96-99.8 %, e.g. Ethanol p.a. (e.g. 9065.1)
Distilled, sterilized H2O
RNAse A, stock solution 100 mg/ml (e.g. 7156.1, 100 mg solubilized in 1 ml distilled
water, made DNAse-free by cooking)
PBS sterile, diluted to 1x (e.g. from 1058.1, Roti®-Stock 10 x PBS)
3. Application
Roti®-Prep-Blood Genomic DNA MINI is designed for isolation of high-purity genomic DNA
from whole blood. Spin column based preparation allows elution in a small volume of lowsalt 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 30 µg gDNA per preparation from common blood
collection systems up to 200 µl or up to 400 µl. If smaller volumes of blood are used, apply
sterile PBS up to 200 µl final sample volume.
The kit co-purifies DNA and RNA, if both are present in the sample. In case RNA-free genomic
DNA is required, RNase A has to be added to the sample after lysis.
The Roti®-Prep-Blood Genomic DNA MINI is not for use with cell-free body fluids such as
cerebrospinal fluid, serum, plasma or urine, tissue or stool samples. The kit performance has
not been evaluated with buffy coat, cultured or isolated cells, swabs, dried blood spots and
viral DNA. The kit is also not specified for the isolation and purification of fungal, bacterial or
parasite nucleic acids.
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Roti®-Prep Blood Genomic DNA MINI
Carl Roth GmbH + Co. KG
The kit is intended for use by professional users and does not provide a diagnostic result. It
is the sole responsibility of the user to use and validate the kit in conjunction with all
required downstream in vitro assays.
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.
Repetition of elution (final step) often enhances the recovery rate overall. However, even so
often, concentration of the recovered gDNA is reduced in parallel, since the whole amount
of buffer used for elution is higher. Elution is 100 µl Elution Buffer EB, followed by another
elution step in 100 µl Elution Buffer EB, may be recommended.
Store the extracted DNA at +4 °C. For long time storage placing at -20 °C 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 blood to be extracted.
Before start, be sure to
- Add Ethanol (96-99.8 %, not incl. in this kit) to the Washing Buffer WSH as follows:
8620.1 (10 Preps): 18 ml (20 ml final vol.)
8620.2 (50 Preps): 90 ml (100 ml final vol.)
8620.3 (250 Preps): 162 ml to each bottle (2x 180 ml final vol.)
Mix thoroughly and keep the bottle always firmly closed!
- Dissolve Proteinase K by addition of distilled H2O as follows:
8620.1 (10 Preps): 0.3 ml
8620.2 (50 Preps): 1.5 ml
8620.3 (250 Preps): 1.5 ml to each tube
Mix thoroughly!
- Heat one thermal mixer or water bath to 60 °C.
- Recommended: heat the needed amount of Elution Buffer EB to 60°C. The final elution step
with heated Elution Buffer will increase the DNA yield!
Please note: Centrifugation steps should be carried out at room temperature
4. 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.
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Carl Roth GmbH + Co. KG
Roti®-Prep Blood Genomic DNA MINI
4.1 Workflow
This workflow describes preparation of gDNA from blood samples of 200 µl. For up-scaling to
up to 400 µl see below.
Step (RT = room temperature)
done
1. Lysis
Transfer 200 µl of the blood sample into a 1.5 ml reaction tube. If the sample is less than
200 µl, add 1x PBS up to 200 µl.
Add 200 µl Lysis Buffer LSN and 20 µl Proteinase K solution. Mix vigorously by pulsed
vortexing for 10 sec.
Incubate for 10 mins at 60 °C under constant agitation.*
Optional: If RNA-free gDNA is required, add 4 µl of RNase A stock solution (100 mg/ml).
Vortex shortly and incubate for 5 additional min at RT.
Centrifuge at full speed for approx. 5 sec. in order to spin down condensed water from
the lid.
Add 350 µl Binding Buffer BR to the lysed sample. Mix carefully by pipetting up and
down.**
2. Column Loading
Place each Spin Column into a 2 ml collection tube
Apply the mix of lysed sample/Binding Buffer BR to the spin column.
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through.***
3. Column Washing
Add 400 µl of Washing Buffer WSO to the Spin Column
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through***
Add 600 µl of Washing Buffer WSH to the Spin Column
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through***
Add 600 µl of Washing Buffer WSH to the Spin Column
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through***
Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 3 mins/RT in order to
remove residual ethanol.
4. Elution
Place the Spin Column into a clean 1.5 ml elution tube
Add 200 µl prewarmed Elution Buffer EB to the centre of the membrane****
Incubate for 2 mins at room temperature
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT to elute DNA
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Roti®-Prep Blood Genomic DNA MINI
Carl Roth GmbH + Co. KG
* We recommend using a shaking platform or thermomixer for continuous agitation of the sample.
Alternatively, vortex the sample 3-4 times during incubation. Agitation is vital for a decent recovery rate.
** Do not vortex the sample at this point! Vortexing will lead to reduced yield of DNA.
*** If the solution has not completely passed through the spin column, centrifuge again at higher speed or
prolong the centrifugation time.
*** Elution with lower volumes of Elution Buffer (e.g. 100 µl Elution Buffer only) increases the final
concentration of DNA. Repetition of the elution step often increases the yield of gDNA overall, but may reduce
the concentration after pooling of the fractions.
4.2 Up-scaling to up to 400 µl blood sample
In case samples of up to 400 µl blood have to be used as source, the following up-scaling for
Lysis has to be applied.
Sample vol.
Lysis Buffer LSN
Proteinase K sol.
RNAse A sol.
Binding Buffer BR
Tube size
200 µl
200 µl
20 µl
4µl
350 µl
1.5 ml
250 µl
250 µl
22 µl
4 µl
440 µl
1.5 ml
300 µl
300 µl
25 µl
4 µl
525 µl
2 ml
400 µl
400 µl
30 µl
4 µl
700 µl
2 ml
In most of these cases, the volume resulting from step 1. Lysis is too big for application to
the spin column in one step. Hence, Column Loading is performed in two steps:
After lysis has been completed, approx. half of the lysed sample / Binding Buffer BR mix is
added to the centre of the spin column membrane. After centrifugation, the second half of
the mix is filtered through the membrane.
Step 3. Column Washing, and step 4. Elution, are performed without any changes in the
amounts given for isolation from 200 µl blood.
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Carl Roth GmbH + Co. KG
Roti®-Prep Blood Genomic DNA MINI
Up-scaling protocol for your specific data
Step (RT = room temperature)
done
1. Lysis
Transfer .............. µl of the blood sample into a 1.5 ml reaction tube.
Add .............. µl Lysis Buffer LSN and .............. µl Proteinase K solution. Mix vigorously by
pulsed vortexing for 10 sec.
Incubate for 10 mins at 60 °C under constant agitation.*
Optional: If RNA-free gDNA is required, add 4 µl of RNase A stock solution (100 mg/ml).
Vortex shortly and incubate for 5 additional min at RT.
Centrifuge at full speed for approx. 5 sec.
Add .............. µl Binding Buffer BR to the lysed sample. Mix carefully by pipetting up and
down.**
2. Column Loading
Place each Spin Column into a 2 ml collection tube
Apply approx. half of the mix of lysed sample/Binding Buffer BR to the spin column.
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through.***
Apply residual mix of lysed sample/Binding Buffer BR to the spin column.
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through.***
3. Column Washing
Add 400 µl of Washing Buffer WSO to the Spin Column
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through***
Add 600 µl of Washing Buffer WSH to the Spin Column
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through***
Add 600 µl of Washing Buffer WSH to the Spin Column
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through***
Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 3 mins/RT in order to
remove residual ethanol.
4. Elution
Place the Spin Column into a clean 1.5 ml elution tube
Add 200 µl prewarmed Elution Buffer EB to the centre of the membrane****
Incubate for 2 mins at room temperature
Centrifuge at 11.000 g (or 12.000 rpm) for 1 min/RT to elute DNA
* We recommend using a shaking platform or thermomixer for continuous agitation of the sample.
Alternatively, vortex the sample 3-4 times during incubation. Agitation is vital for a decent recovery rate.
** Do not vortex the sample at this point! Vortexing will lead to reduced yield of DNA.
*** If the solution has not completely passed through the spin column, centrifuge again at higher speed or
prolong the centrifugation time.
*** Elution with lower volumes of Elution Buffer (e.g. 100 µl Elution Buffer only) increases the final
concentration of DNA. Repetition of the elution step often increases the yield of gDNA overall, but may reduce
the concentration after pooling of the fractions.
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Roti®-Prep Blood Genomic DNA MINI
Carl Roth GmbH + Co. KG
5. Trouble Shooting
Problem / probable cause
Clogged spin filter
Comments and suggestions
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.
Low recovery
Insufficient lysis
Insufficient mixing with Binding Buffer
BR
Low amount of recovered gDNA.
Low concentration of eluted gDNA
Degraded or sheared DNA
Incorrect storage of starting material.
Old material
RNA contamination
Unsufficient RNAse digestion during
lysis
See above
Mix sample very well with Binding Buffer BR by
pipetting prior to transfer of the sample onto the
Spin Filter
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.
Reduce the volume of Elution Buffer used.
Optionally: prolong elution / incubation time.
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.
Use fresh material. Check and improve storage
conditions.
Add RNAse A after lysis.
Check storage conditions and usage of RNAse A.
Optionally replace RNAse A by a fresh lot.
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Carl Roth GmbH + Co. KG
Roti®-Prep Blood Genomic DNA MINI
Your notes:
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Roti®-Prep Blood Genomic DNA MINI
Carl Roth GmbH + Co. KG
Ordering information:
(for detailed kit content see Table under 1.)
Product
number
Product
Content
Amount
8620.1
Roti®-Prep Blood Genomic DNA MINI
1 Test-Kit
10 Preps
8620.2
Roti®-Prep Blood Genomic DNA MINI
1 Kit
50 Preps
8620.3
Roti®-Prep Blood Genomic DNA MINI
1 Maxi-Kit
250 Preps
To place your order, please contact us:
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Phone (Germany): 0800/56 99 000
Phone (international): +49 (0)721/5606-0
Fax (Germany and international): +49 (0)721/5606-149
E-Mail (Germany): bestellungen@carlroth.de
E-Mail (international): info@carlroth.com
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Carl Roth GmbH + Co. KG
Phone: +49 (0)721/5606-0
Schoemperlenstraße 3-5
Fax:
76185 Karlsruhe, Germany
Email: info@carlroth.com
+49 (0)721/5606-149