Contents - Biomiga
Contents
Introduction.........................................................................................
2
Storage and Stability...........................................................................
2
Safety Information..............................................................................
Kit Contents.........................................................................................
3
Before Starting.....................................................................................
3
Protocol for Dried Body Fluid Samples..............................................
4
Protocol for DNA Isolation from Sperm.............................................
6
Protocol for Buccal Swabs..................................................................
8
Protocol for Bacterial DNA From Biological Fluids..........................
9
Protocol for Eye, Nasal and Other Swabs...........................................
9
Protocol for Saliva...............................................................................
10
Protocol for Hair, Nails and Feathers..................................................
10
Vacuum/Spin Protocol........................................................................
11
Determination of Yield and Quality...................................................
12
Trouble Shooting Guide......................................................................
13
Related Products…………………………………………………….
15
Limited Use and Warranty…………………………………………..
16
Page 1
Biomiga EZgene
TM
Forensic gDNA Kit
Introduction
The EZgeneTM Forensic gDNA Purification Kit provides a rapid and easy method
for the isolation of genomic DNA from forensic samples, such as dry blood, buccal
swabs, and sperm for consistent PCR and Southern analysis. This kit can also be
used for the preparation of genomic DNA from mouse tail snips, whole blood,
buffy coat, serum, and plasma. No phenol/chloroform extractions and isopropanol
or ethanol precipitation are needed. DNA purified using this method is ready for
downstream applications such as PCR, Southern blotting, and restriction digestion.
In this procedure, samples are first lysed and applied to the spin column that DNA
binds. While cellular debris, hemoglobin, and other proteins are effectively washed
away by DNA Wash Buffer, pure DNA is eluted in sterile deionized water or
elution buffer. Each ezBind column can bind approximately 100 μg DNA.
Storage and Stability
Once reconstituted, Protease K must be stored at -20°C. All other components can
be stored at 22-25°C.Under these conditions, performance of all components of the
kit are guaranteed at least 12 months from the date of purchase. Under cool ambient
conditions, a precipitate may form in the Buffer BL; heat the bottle at 37°C to
dissolve the precipitate before use.
Safety Information
Buffer BL contains acidic acid and chaotropic salts, which may form reactive
compounds when combines with bleach. Do not add bleach or acidic solutions
directly to the preparation waste. Ware gloves and protective eyeware when
handling this solution.
Page 2
Biomiga EZgeneTM Forensic gDNA Kit
Kit Contents
Product
GD2512-00
GD2512-01
GD2512-02
Preps
4
50
250
DNA Columns
4
50
250
2 mL Collection Tubes
8
100
500
Buffer BL
3 mL
30 mL
150 mL
Buffer TL
3 mL
20 mL
100 mL
Buffer KB
3 mL
28 mL
135 mL
DNA Wash Buffer
2 mL
15 mL
3 x 24 mL
Elution Buffer
2 mL
15 mL
60 mL
Protease K
3 mg
30 mg
5 x 30 mg
1
1
1
User Manual
For isolation gDNA from sperm
Product
10x Buffer A
Buffer B
GD2512-00
5 mL
1 mL
GD2512-01
50 mL
15 mL
GD2512-02
250 mL
70 mL
Note: The kit is supplied with enough buffers for the standard protocol. However,
due to increased volumes called for in some protocols (such as the buccal swab
protocol), extra buffers can be purchased separately from Biomiga. See product
inforamtion on our website or call customer service for price information.
Before Starting
Important:

Reconstitute Protease K in 110 μL (GD2512-00) or 1.3 mL (GD2512-01) or 5
x 1.3 mL (GD2512-02) Elution Buffer. Vortex the vial and spin the vial
briefly prior to use.

Dilute DNA Wash Buffer with absolute ethanol as follows:
 GD2512-00 Add 8 mL ethanol
 GD2512-01 Add 60 mL ethanol
 GD2512-02 Add 96 mL ethanol/bottle

Prepare DL-Dithiothreitol (DTT) for Hair, Nails and Feathers protocol.
Page 3
Biomiga EZgene
TM
Forensic gDNA Kit
Protocol for Isolation of DNA from Dried Blood, Body
Fluids and Sperm Spots
Dried blood, body fluids, and sperm samples on filter paper can be processed using
the following method. Use Specimen paper or blot paper for spotting samples as
the filter paper disintegrates DNA when aqueous buffers are added.
1. Punch out the sample spot from the filter paper. (Use less than 200 μL of blood
each spot). Tear or cut filter into small pieces and place into a microfuge tube.
Note: Use 3 mm diameter cycle puncher for each sample.
2. Add 200 μL Buffer TL and incubate at 55 °C for 15-20 min. Mix well by
vortexing 3-5 times during incubation.
3. Add 25 μL Protease K solution and mix well by votexing. Incubate for at 60°C
for 30-60 min with occasional mixing. Spin the sample briefly to collect
droplets from the cap.
4. Add 225 μL Buffer BL and mix well by vortexing. Incubate at 60°C for 10 min.
Spin the sample briefly to collect an droplets from the cap.
5. Add 225 μL absolute ethanol and mix thoroughly by vortexing. Briefly
centrifuge to remove any droplets inside the tube.
6. Apply the entire sample into a DNA column, including any precipitate that may
have formed. Centrifuge at 10,000 rpm for 1 min. Discard collection tube and
flow-through liquid.
7. Transfer the column into a new collection tube and add 500 μL of Buffer KB
into column. Centrifuge at 10,000 rpm for 30s. Dispose of flow through liquid
and re-use the collection tube.
8. Add 650 μL of DNA Wash Buffer. Centrifuge at 10,000 rpm for 30s. Discard
the flow-through liquid and put the column back to the collection tube.
9. Add 650 μL of DNA Wash Buffer and centrifuge as above. Discard flowthrough and put the column, with the lid open, to a new collection tube.
Page 4
Biomiga EZgeneTM Forensic gDNA Kit
10. Centrifuge at the column at 13,000 rpm to dry the column. This step is critical
for removing residual ethanol that might interfere with DNA yield and purity.
11. Place the column into a sterile1.5 mL tube and add 100 μL of preheated (70 °C)
Elution Buffer. Incubate the tube at 70°C for 3 minutes.
12. Centrifuge at 10,000 rpm for 1 min to elute the DNA.
Note: Add the eluted DNA back to the column for a second elution
will yield another 20% of DNA bound.
Note: Incubation at 70°C rather than at room temperature will give a modest
increase in DNA yield per elution.
Note: Blood spots from finger pricks usually contain no more than 50 μL blood
and yield approximately 0.5 -1 μg DNA. This is sufficient for PCR analysis.
Page 5
Biomiga EZgene
TM
Forensic gDNA Kit
Protocol for Isolation of Genomic DNA from Sperm
This protocol can be used for fresh or frozen semen samples with equal efficiency.
Frozen samples must to be thawed thoroughly before use. Note that lysis time will
vary depending on the size and density of the source material.
Dilute the following buffer before starting:
Buffer A: Dilute 10× Buffer A to 1× Buffer A with ddH2O before use.
Buffer B: Add ß-mercaptoethanol to final percentage of 2% (v/v).
1. Add 50-250 μL of sperm to 10 mL of Buffer A in a glass (Corex) centrifuge
tube. Vortex for 10 seconds at full speed. Only use Corex tubes to prevent
attachment of the sperm cells to the tube walls.
2. Centrifuge for 10 min at 4,000 rpm (2500 x g).
3. Carefully remove the supernatant leaving 1 mL of pellet and Buffer A.
4. Vortex for 5s and centrifuge at full speed to collect any sample adhering to the
walls of tubes. Transfer the sample to a 2.0 mL tube.
5. Add 0.5 mL of Buffer A to the Corex tube and vortex for 30s and centrifuge at
full speed to collect any sample adhering to the walls of the tube. Transfer
sample to a 2 mL microfuge tube.
6. Centrifuge for 2 min at full speed.
7. Carefully remove the supernatant without disturbing the semen pellet.
8. Resuspend pellet in 200 μL of Buffer B.
9. Add 25 μL Proteinase K (25 mg/mL) and incubate for 2 hours at 60°.
10. Invert the tube occasionally to disperse the sample or place on a rocking
platform.
Page 6
Biomiga EZgeneTM Forensic gDNA Kit
11. Add 250 μL Buffer BL and 260 μL ethanol to the sample and mix by
vortexing.
12. Go to step 6 on page 4 and follow the standard protocol to carry out the DNA
purification.
Page 7
Biomiga EZgene
TM
Forensic gDNA Kit
Protocol for Isolation of Genomic DNA from Buccal
Swabs
Typical yields from cotton or C.E.P. swabs are typically around 0.5 - 3 μg DNA.
1. Follow standard protocol; scrape the swabs firmly against the inside of cheek 510 times. Air-dry or vacuum the swabs for 1-2 hours after collection. The
person providing the sample should not eat or drink for at least 30 min prior to
the sample collection.
2. Remove and transfer the buccal swab into a 2.0 mL tube and add to the tube.
3. Add 525 μL PBS, 25 μL Protease K solution and 525 μL Buffer BL to the
sample. Mix immediately by votexing for 30s. Incubate 30 min at 60 °C with
occasional mixing. Briefly centrifuge to remove any droplets from inside the lid.
4. Add 525 μL absolute ethanol and mix thoroughly by vortexing. Briefly
centrifuge to collect any droplets from the lid.
5. Carefully apply 600 μL of the mixture into the column. Centrifuge at 10,000
rpm for 30s. Discard the flow-through liquid and put the column back to the
collection tube.
6. Carefully apply the remaining mixture to the column and centrifuge at 10,000
rpm for 30s. Discard the flow-through liquid and collection tube.
7. Put the column into another collection tube, Add 650 μL of DNA Wash Buffer
and centrifuge at 10,000 rpm for 1 min. Discard the flow-through liquid and put
the column back to the collection tube.
8. Add 650 μL of DNA Wash Buffer and centrifuge as above. Discard the flowthrough liquid and collection tube. Put the column, with the lid open, to a new
collection tube.
9. Centrifuge at 13,000 rpm for 2 min to dry the column. This step is critical for
removal of residual ethanol that might result in decreased DNA yield and purity.
Page 8
Biomiga EZgeneTM Forensic gDNA Kit
10. Place the column into a nuclease-free 1.5 mL microfuge tube and add 100 μL
of preheated (70°C) Elution Buffer. Allow tube to incubate at 70°C for 3 min.
11. Centrifuge at 10,000 rpm for 1 min to elute DNA.
Note: Add the eluted DNA back to the column for a second elution
will yield another 20% of DNA bound.
Note: Incubation at 70°C rather than at room temperature will give a modest
increase in DNA yield.
Protocol for Isolation of Bacterial gDNA from
Biological Fluids
1. Pellet bacteria by centrifuging 10 min at 8,000 rpm.
2. Resuspend the pellet with 200 μL Buffer TL.
3. Follow the standard protocol (Page 4) from Step 3.
Protocol for Isolation of Genomic DNA from Eye, Nasal,
and Other Swabs
1. Collect the sample and add 2 mL PBS. Incubate 2-3 hours at 30°C.
2. Pellet bacteria by centrifuging 10 min at 8,000 rpm.
3. Resuspend bacterial pellet with 200 μL Buffer TL.
4. Follow the standard protocol (Page 4) from Step 3.
Page 9
Biomiga EZgene
TM
Forensic gDNA Kit
Protocol for Isolation of DNA from Salvia
1. Add1.5 mL saliva to a 15 mL centrifuge tube contains 6 mL PBS.
2. Vortex to mix well.
3. Centrifuge at 2000 x g for 5 min. Discard the supernatant and ressuspend the
pellet in 180 μL PBS.
4. Transfer the sample to a new 1.5 mL centrifuge tube.
5. Add 25 μL Protease K solution, 200 μL Buffer BL. Add 20 μL RNase A
solution if RNA-free DNA is desired. Mix throughly by vortexing for 10s.
Briefly centrifuge to collect any droplets from the lid. Incubate 15 min for 70°C
with occasional mixing.
6. Add 200 μL absolute ethanol and mix throughly by voretxing.
7. Go to Step 6 on page 4 and carry out the purification procedure.
Protocol for Isolation of DNA from Hair, Nails and
Feathers
1. Cut the sample into small pieces (0.5-1 cm) and transfer to a 1.5 mL centrifuge
tube.
Note: For hair, cut from base of hair for feathers: select the primary feathers.
(Large birds, secondary tail or breast feather can be used).
2. Add 250 μL TL Buffer, 25 μL Protease K and 20 μL 1 M DTT. Mix
thoroughly by vortexing. Incubate 30 min at 60°C with occasional mixing.
3. Add 250 μL Buffer BL to the sample, mix throughly by vortexing. Add 250 μL
absolute ethanol to the sample, mix thoroughly by vortexing.
4. Follow the standard protocol from Step 6 on Page 4.
Page 10
Biomiga EZgeneTM Forensic gDNA Kit
Vacuum/Spin Protocol
Go through the previous sections of this manual before using this protocol.
1. Prepare samples by following the standard protocol in previous sections (Steps
1-5).
2. Prepare the vacuum manifold according to manufacturer’s instructions and
connect the column to the manifold.
3. Load the sample/Buffer BL/ethanol mixture into the column.
4. Switch on vacuum source to draw the sample through the column; then turn off
the vacuum.
5. Wash the column by adding 650 μL DNA Wash Buffer. Draw the DNA Wash
Buffer through the column by turning on the vacuum source. Repeat this step
with another 650 μL DNA Wash Buffer.
6. Transfer the column, with the lid open, to a 2 mL collection tube. Centrifuge at
13,000 rpm for 1 min to dry the column. It is critical to remove the residual
ethanol completely for optimal DNA yield and purity.
7. Place the column in a nuclease-free 1.5 mL microcentrifuge tube and add 50100 μL Elution Buffer or ddH2O. Allow standing for 1-2 min, and then
centrifuge 1 min to elute DNA.
Note: Add the eluted DNA back to the column for a second elution
will yield another 20% of DNA bound.
Note: Incubation at 70°C rather than at room temperature will give a
modest increase in DNA yield.
Page 11
Biomiga EZgene
TM
Forensic gDNA Kit
Determination of Yield and Quality
The total DNA yield can be determined by,
[DNA] = (Absorbance260) x (0.05 μg/ μL) x (Dilution factor)
The quality of DNA can be evaluated by OD260/280. A ratio of (A260/A280) of 1.8-1.9
corresponds to 90%-95% purity. Expected yields vary with both amount, and type
of tissue used. 30 mg of fresh tissue will yield approximately10-40 μg DNA with
two elutions (each 100 μL).
Page 12
Biomiga EZgeneTM Forensic gDNA Kit
Trouble Shooting Guide
Problem
Possible Cause
Forgot
ethanol
Colored
residue in
column
After
washing
Column
clogged
to
Suggestions
add Before applying sample to column, both Buffer BL
and ethanol must be added. See protocol above.
Forgot to add
ethanol to DAN
Wash Buffer
Dilute DNA Wash Buffer with the indicated
volume of absolute ethanol before use.
Incomplete lysis
Buffer BL is viscous and the sample must be
due to improper
vortexed thoroughly.
mixing with Buffer
BL.
No ethanol added
to DNA Wash
Buffer
Concentrate.
Dilute DNA Wash Buffer with the indicated
volume of absolute ethanol before use.
Incomplete lysis
Extend incubation time of lysis with Buffer TL
and protease. Add the correct volume of Buffer BL
and incubate for specified time at 70℃. It may be
necessary to extend incubation time by 10 min.
Sample too large
If using more than 30 mg tissue, increase volumes
of Proteinase K, Buffer TL, Buffer BL, and
ethanol. Pass aliquots of lysate through one
column successively.
Sample too viscous Divide sample into multiple tubes, adjust volume
to 250 μL with 10 mM Tris-HCl.
Low
DNA
yield
Clogged column
See above
Poor sample
release from
collection paper
Incubate the OB specimen collection paper longer
in TL buffer. Shake the tubes frequently.
Poor elution
Repeat elution or increase elution volume.
Incubation of column at 70℃ for 5 min with
Elution Buffer may increase yields.
Improper washing
DNA Wash Buffer Concentrate must be diluted
with absolute (100%) ethanol as specified on Page
3 before use.
Page 13
Biomiga EZgene
TM
Forensic gDNA Kit
Trouble Shooting Guide (Continue from page 13)
Problems
Possible Cause
Suggestions
Extended
Resin from the column may be present in eluate.
centrifugation
Avoid centrifugation at speeds higher than
during elution step. specified. The material can be removed from the
eluate by centrifugation — it will not interfere
with PCR or restriction digests.
Poor cell lysis due Repeat the procedure, this time making sere to
to incomplete
vortex the sample with Buffer BL immediately
mixing with Buffer and completely.
BL
A260 /A280
ratio lower
Incomplete cell
than 1.7
lysis or protein
degradation due to
insufficient
incubation.
Increase incubation time with Buffer TL and
protease. Ensure that no visible pieces of tissue
remain.
Samples are rich in After applying to column, wash with 300 μL of a
protein.
1:1 mixture of Buffer BL and ethanol and then
with DNA Wash Buffer.
Poor cell lysis due Mix thoroughly with Buffer BL prior to loading
No DNA
to improper mixing ezBind column.
eluted
with Buffer BL.
Page 14
Biomiga EZgeneTM Forensic gDNA Kit
Related EZgeneTM Products
Catalog #
Product Name
Preps
Price $
GD2211-01
Tissue gDNA kit
50
90.00
GD2211-02
Tissue gDNA kit
250
420.00
GD2814-01
96-well tissue gDNA kit
4x96
680.00
GD2814-02
96-well tissue gDNA kit
20x96
3000.00
GD2311-01
Blood gDNA mini kit
50
90.00
GD2311-02
Blood gDNA mini kit
250
420.00
GD2312-01
Blood gDNA midi kit
10
90.00
GD2312-02
Blood gDNA midi kit
25
200.00
GD2314-01
Blood gDNA maxi kit
10
160.00
GD2314-02
Blood gDNA maxi kit
25
360.00
GD2815-01
96-well blood gDNA kit
4x96
680.00
GD2815-02
96-well blood gDNA kit
20x96
3000.00
GD2411-01
Bacterial gDNA kit
50
110.00
GD2411-02
Bacterial gDNA kit
250
495.00
GD2412-01
Soil gDNA kit
50
90.00
GD2412-02
Soil gDNA kit
250
420.00
GD2413-01
Insect gDNA kit
50
90.00
GD2413-02
Insect gDNA kit
250
420.00
GD2414-01
Mullusc gDNA kit
50
90.00
GD2414-02
Mullusc gDNA kit
250
420.00
GD2415-01
Yeast gDNA kit
50
90.00
GD2415-02
Yeast gDNA kit
250
420.00
GD2416-01
Fungal gDNA kit
50
90.00
GD2416-02
Fungal gDNA kit
250
420.00
GD2512-01
Forensic gDNA purification kit
50
85.00
GD2512-02
Forensic gDNA purification kit
250
380.00
Page 15
Biomiga EZgene
TM
Forensic gDNA Kit
Limited Use and Warranty
This product is intended for in vitro research use only. Not for use in human.
This product is warranted to perform as described in its labeling and in Biomiga’s
literature when used in accordance with instructions. No other warranties of any
kind, express or implied, including, without limitation, implied warranties of
merchantability or fitness for a particular purpose, are provided by Biomiga.
Biomiga’s sole obligation and purchaser’s exclusive remedy for breach of this
warranty shall be, at the option of Biomiga, to replace the products, Biomiga shall
have no liability for any direct, indirect, consequential, or incidental damage arising
out of the use, the results of use, or the inability to use it product.
For technology support or learn more product information, please visit our
website at www.biomiga.com or contact us at (858) 603-3219.
FOR REACHER USE ONLY
Page 16
Biomiga EZgeneTM Forensic gDNA Kit
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