Simply Blood DNA Mini Kit Manual SG-P-023-1

Simply Blood DNA Mini Kit
Manual
SG-P-023-1
Contents
Contents
1
General Information
Introduction . . . . . . . .
Intended use . . . . . . .
Product specifications . . .
List of Components . . . .
Storage conditions . . . .
Safety information . . . . .
Hazard Identification
1
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2
2
2
2
3
3
3
4
2
Additional Requirements
Required equipment and additives . . . . . . . . . . . . . . . . . . . . . . . . .
Required components not included . . . . . . . . . . . . . . . . . . . . . . . . .
5
5
5
3
Protocol
Recommended steps before use . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
6
6
4
Troubleshooting
8
5
Warranties and Quality Control
9
6
Technical support
Legal Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
10
7
Your notes
11
Simply Blood DNA Mini Kit
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Page 1
1
General Information
Introduction
The Simply Blood DNA Mini Kit provides a very fast and simple method for the preparation of total DNA from whole blood samples.
The extraction procedure is based on binding nucleic acids to the surface of a Spin Filter
membrane and consists of four steps. During the first step, cells and nuclei are lysed.
Subsequently, the DNA is bound to a Spin Filter membrane, washed and, finally, eluted.
Intended use
The kit is designed for the preparation of DNA from whole blood samples. It is suitable to
process up to 400 µl of fresh or frozen whole blood, stabilized with EDTA or citrate. The
extracted DNA can be used for a wide range of different molecular biology downstream
applications, including PCR, restriction digestion, cloning and sequencing.
B
For Research Use Only — Not for use in diagnostic procedures.
Product specifications
Starting Material
Fresh or frozen whole blood (200 or 400 µl), stabilized with EDTA or citrate
Time for preparation
approx. 30 minutes
Binding capacity
more than 60 µg genomic DNA
Expected Yield
at least 30 µg genomic DNA, depending on initial sample volume
Purity indicator
Ratio A260 :A280 between 1.7 and 2.0
Order number
SG-P-023-1 (50 reactions)
Manual version
SG-P-023-1-1
Page 2
Simply Blood DNA Mini Kit
List of Components
Component
Quantity
Storage
Cat No
Binding Solution C
40 ml
15 - 25°C
SG-C-031-1
Collection Tube B
250 pcs
15 - 25°C
SG-C-009-1
Elution Buffer A
6x 2 ml
15 - 25°C
SG-C-063-1
Elution Tube A
50 pcs
15 - 25°C
SG-C-038-1
Lysis Solution D
25 ml
15 - 25°C
SG-C-055-1
Proteinase K II
2 pcs
4°C (if dissolved: -20°C)
SG-C-022-1
Spin Filter Red A
50 pcs
15 - 25°C
SG-C-018-1
Washing Solution A
25 ml
15 - 25°C
SG-C-044-1
Washing Solution G
8 ml
15 - 25°C
SG-C-045-1
Storage conditions
All components of the Simply Blood DNA Mini Kit except for the Proteinase K II should
be stored dry at room temperature (15 to 25°C).
Lyophilized Proteinase K should be stored at 4°C. Dissolved Proteinase K should be
stored at -20°C. Repeated freezing and thawing should be avoided. Allocation of the solution into aliquots is recommended.
If there are any precipitates within the provided solutions solve these precipitates by careful
warming. Do not use the kit after the expiration date.
Safety information
All reagents in this kit have to be used for the intended use described above only. The kit
should be used by authorized staff only. It is strongly recommended to follow the instruction for use. Inappropriate handling or use of the kit or non-observance of the instructions
for use can increase the danger for your health and reduce the reliability of results. The
reagents should be stored inside the original vessels at the given storage temperatures as
indicated on the Fact Sheet. Single components of different lots and consumables should
not be exchanged. The expiry dates have to be considered. Please pay attention to the
regulations for the prevention of accidents in laboratories. In particular, the following safety
precautions have to be considered:
B
Don’t eat, drink or smoke! Always wear protective clothing, safety glasses and
gloves! Do not add bleach or acidic components to the waste after sample
preparation! All liquid waste must be considered as potentially infectious and
must be handled and discarded according to local safety regulation!
Simply Blood DNA Mini Kit
Page 3
For more information, please read the Fact Sheet and ask for the material safety data
sheets (MSDS’s). Emergency medical information in English and German can be obtained 24 hours a day from the Poison Information Center in Freiburg (Germany), Phone:
+49 761 19240.
Hazard Identification
Please read carefully the following hazard identification information that classifies the substances or mixtures contained in this product.
Proteinase K II
Signal word
DANGER
Pictograms
Hazard statement(s)
H315
Causes skin irritation.
H319
Causes serious eye irritation.
H334
May cause allergy or asthma symptoms or breathing difficulties if inhaled.
H335
May cause respiratory irritation.
Precautionary statement(s)
P261
Avoid breathing dust/fume/gas/mist/vapours/spray.
P305 + P351 + P338
IF IN EYES: Rinse cautiously with water for several minutes. Remove
contact lenses, if present and easy to do. Continue rinsing.
P342 + P311
If experiencing respiratory symptoms: Call a POISON CENTER / doctor / ...
R-phrase(s)
R32
Contact with acids liberates very toxic gas.
R36/37/38
Irritating to eyes, respiratory system and skin.
S-phrase(s)
S22
Do not breathe dust.
S24
Avoid contact with the skin.
S26
In case of contact with eyes, rinse immediately with plenty of water
and seek medical advice.
S36/37
Wear suitable protective clothing and gloves.
Page 4
Simply Blood DNA Mini Kit
2
Additional Requirements
Required equipment and additives
2 Microcentrifuge
2 Thermomixer
2 Vortexer
2 Variable pipettes for 10 µl – 1 000 µl
2 Sterile pipette tips with protection against contamination
Required components not included
2 1.5 ml reaction tubes
2 optionally RNase A (100 mg/ml)
2 96 - 99.8 % ethanol
2 ddH2 O
Simply Blood DNA Mini Kit
Page 5
3
Protocol
This protocol is valid for the preparation of total DNA of 200 or 400 µl whole blood (fresh
or frozen, stabilized with EDTA or citrate).
Recommended steps before use
Before first use:
2 Washing Solution G: Add 72 ml of ethanol (96 – 99.8 %)
2 Proteinase K II: Add 1.5 ml of ddH2 O, mix thoroughly and store in aliquots at -20°C
Before use:
• Thermomixer: Preheat to 60°C
• Elution Buffer A: Preheat to 60°C
• Make sure that all other components have room temperature
Procedure
1. Add 200 / 400 µl of blood into a 1.5 ml reaction tube.
Note: If the blood volume is less than 200 / 400 µl, add the appropriate volume of
PBS.
2. Add 200 / 400 µl of Lysis Solution D and 20 / 30 µl Proteinase K II to the sample.
Mix by pulsed vortexing for 10 seconds.
3. Incubate at 60°C for 10 minutes at 550 rpm.
Note: If required, add 4 µl of an RNase A stock solution (100 mg/ml) to remove
RNA from sample, vortex shortly and incubate for 5 minutes at room temperature.
4. Add 350 / 700 µl of Binding Solution C to the lysed sample. Mix carefully by
pipetting up and down 3 – 4 times. Don’t vortex!
5. Place the Spin Filter Red A into a Collection Tube B.
Page 6
Simply Blood DNA Mini Kit
6. Apply the sample to the Spin Filter Red A and close the cap. Centrifuge at 11 000 x g
for 1 minute.
Attention: The 400 µl blood - protocol needs to divide the sample into two parts.
First add 750 µl of the sample to the Spin Filter Red A and close the cap. Centrifuge
at 11 000 x g for 1 minute. Place the Spin Filter Red A into a new Collection Tube B
and discard the used Collection Tube B. Add the residual sample to the Spin Filter
Red A. Close the cap and centrifuge at 11 000 x g for 1 minute.
7. Place the Spin Filter Red A into a new Collection Tube B and discard the used
Collection Tube B.
8. Add 400 µl of Washing Solution A and close the cap. Centrifuge at 11 000 x g for
1 minute.
9. Place the Spin Filter Red A into a new Collection Tube B and discard the used
Collection Tube B.
10. Add 600 µl of Washing Solution G and close the cap. Centrifuge at 11 000 x g for
1 minute.
11. Place the Spin Filter Red A into a new Collection Tube B and discard the used
Collection Tube B.
12. Add 600 µl of Washing Solution G and close the cap. Centrifuge at 11 000 x g for
1 minute.
13. Place the Spin Filter Red A into a new Collection Tube B and discard the used
Collection Tube B.
14. Centrifuge at max. speed for 3 minutes (above 11 000 x g) to avoid a carry-over of
ethanol residuals.
15. Place the Spin Filter Red A into the 1.5 ml Elution Tube A.
16. Add 200 µl of Elution Buffer A (preheated to 60°C) and incubate at room temperature for 2 minutes. Centrifuge at 11 000 x g for 1 minute.
Note: To increase the concentration of the extracted DNA, elute in two steps with
preheated Elution Buffer A (e.g. 100 µl + 100 µl) or elute with volumes smaller than
200 µl.
17. Store the extracted DNA at 4°C. Long term storage of DNA should happen at -20°C.
Simply Blood DNA Mini Kit
Page 7
4
Troubleshooting
Problem
Probable Cause
Remedy
Increase lysis time
Slow flow speed through
the Spin Filter Red A
Insufficient lysis
Overloading of Spin Filter reduces yields
Increase centrifugation speed / time
Insufficient lysis
See above
Blood sample may contain too
few white blood cells
Draw new blood sample
Blood sample too old
Storage at 2 to 5°C for more than 5 days
may reduce the yields
Insufficient mixing with Binding
Solution C
Mix carefully by pipetting up and down 3 –
4 times. Don’t vortex!
Poor DNA yield
Incomplete elution
Increase the incubation time with Elution
Buffer A to 5 minutes
Repeat Elution step once again
Increase the Elution Buffer A volume
Too much Elution Buffer A
Elute the DNA with lower Elution Buffer A
volume
Incorrect storage of the sample Avoid thawing and freezing
Degraded or sheared
DNA
Low performance of
downstream applications
RNA contamination
Page 8
Improper collection of the
blood sample
Draw new blood sample
Incorrect storage of the DNA
Avoid thawing and freezing
Salt carry-over
Ensure proper washing
Check Wash Buffers for salt precipitates
Ethanol carry-over
Increase centrifugation time for ethanol
removal
RNA
Digest RNA with RNase
Simply Blood DNA Mini Kit
5
Warranties and Quality Control
Simply Genomics guarantees the correct performance of each kit for applications as described in the instruction for use.
The purchaser must determine the suitability of the product for its particular use.
We reserve the right to change, alter or modify any product to enhance its performance
and design.
If a Simply Genomics product does not meet your expectations or if you are unsatisfied
for any reason with this product, please contact our Technical Assistance.
Simply Blood DNA Mini Kit
Page 9
6
Technical support
If you have any questions regarding any aspects of this product, please do not hesitate to
contact us via
support@simplygenomics.com
or call us at +49 (0) 3641 / 777 888.
Legal Information
This product has been manufactured, distributed and published by:
Moldiax GmbH
Konrad-Zuse-Str. 1
07745 Jena
Germany
Phone: +49 (0) 3641 / 777 333
E-Mail: info@moldiax.de
Internet: www.moldiax.de
© Copyright 2014, Moldiax GmbH
Page 10
Simply Blood DNA Mini Kit
7
Your notes
Simply Blood DNA Mini Kit
Page 11
Page 12
Simply Blood DNA Mini Kit
Moldiax GmbH
Konrad-Zuse-Straße 1
07745 Jena / Germany
Telefon +49 (0) 36 41 / 777 333
Telefax +49 (0) 36 41 / 777 67 333
info@simplygenomics.com
www.simplygenomics.com