EZ-10 SPIN COLUMN GENOMIC DNA

MINIPREPS KIT HANDBOOK

(Bacteria, Plant, Animal, Blood)

Version 4.8 Rev 05/01/2013

 EZ-10 Genomic DNA Kit Handbook

Table of Contents Introduction

Introduction 2 EZ-10 Spin Column Kits provide a fast, simple and efficient method for
Limitations of Use 2 purification of genomic DNA from various sources such as Bacteria, Plant tissue,
Features 2 Animal tissue, Cells and Blood.
By taking the advantage of silica-based DNA purification technology, DNA is
Applications 2 selectively adsorbed in silica-based membrane embeded in EZ-10 Spin Column.
Storage 2 Other components and impurities flow through the column or are washed away
Quality Control 2 during wash steps. Genomic DNA is then eluted off the column and can be readily
EZ-10 Spin Column Genomic DNA Minipreps Kit, Bacteria 3 used in most downstream applications, including restriction enzyme digestion,
Kit Contents 3 PCR, Southern-blotting etc.
The purification procedure using in these kits do not require use of hazardous
Principle 3 compound such as phenol, chloroform, or CsCl. DNA is purified without additional
Procedures for Isolation of Genomic DNA from Cells or Bacteria 3 steps of ethanol precipitation.
Troubleshooting Guide 5 Limitations of Use
EZ-10 Spin Column Genomic DNA Minipreps Kit, Plant 7 These kits are designed for research use only. Purified DNA should not be
Kit Contents 7 used for live animal transfections. It is also not to be used for human diagnostic or
Procedures for Isolation of Genomic DNA from Plants 8 drug production purposes.
Troubleshooting Guide 9 Features
EZ-10 Spin Column Genomic DNA Minipreps Kit, Animal 10 Simple, fast and efficient.
Preparation of high quality genomic DNA from various sources.
Kit Contents 10 High yield and reproducible.
Procedures for Isolation of Genomic DNA from Animal 11 No phenol chloroform extraction or ethanol precipitation required.
For Animal Tissue 11 High capacity up to 10 µg of DNA per column.
For Rodent Tail 12 Applications
For Cultured Animal Cell 13 Obtain up to 10 µg of genomic DNA purification from various sources.
From Paraffin Tissue 14 Storage
EZ-10 Spin Column Genomic DNA Minipreps Kit, Blood 16 All components except Proteinase K could be stored at room temperature.
Kit Contents Proteinase K should be kept at 4ºC for short term or -20ºC for long term storage.
16
Kits are stable for 12 months at room temperature after received. For maximum
Storage of Blood 16 stability, store all contents at 4ºC.
Blood Collection and Treatment 17 Quality Control
Procedures for Extraction Genomic DNA from Blood 17 Each lot of EZ-10 Spin Column kit is tested against predetermined
Troubleshooting Guide 18 specifications to ensure consistent product quality.

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 EZ-10 Genomic DNA Kit Handbook

EZ-10 Spin Column Genomic DNA Minipreps Kit, Bacteria

Kit Contents Spin appropriate number of cells (not exceed 5 x 106 cells) at 2,500 x g (5,000
rpm) for 5 minutes at room temperature. Remove supernatant completely. Wash
BS423, BS624, the cell pellet twice with PBS Buffer (not provided with kit) and resuspend cells in
Component
50 Preps 250 Preps 200 µl cold TE Buffer (not provided with kit), proceed to Step 2.
Digestion Solution (a) 20 ml 100 ml
(2) Cells grown in monolayer
(b)
Wash Solution 12 ml 2 x 30 ml
Aspirate the medium and wash cells with PBS Buffer. Remove PBS and apply
Elution Buffer (c) 5 ml 25 ml trypsin solution to the cells. After cells have become detached, neutralize the
Proteinase K (d) 2 mg 10 mg trypsin with 2 volumes of medium. Centrifuge at 8,000 x g (10,000 rpm) for 5
minutes. Carefully remove supernatant and resuspend pellet in 200 µl TE buffer,
EZ-10 Column (with 2.0-ml Collection Tube) 50 250
and continue with Step 2.
Protocol 1 1
If following steps could not be performed immediately, it is safe to pause
Notes: here and it is recommended to store the lysate at -20 ºC or -80 ºC.
a. Digestion Solution may form precipitates upon storage. Dissolve the Avoid repeated freezing and thawing of stored samples, since this leads
precipitate by warming the solution at 37ºC if necessary. to reduced DNA size and yield.

b. Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS423,
or 120 ml of 100% ethanol to 30 ml Wash Solution for BS624. For other
volumes of Wash Solution, simply add ethanol to make a 4:1 ratio (volume
of added ethanol:volume of Wash Solution = 4:1).
c. The recipe of Elution Buffer is 2.0 mM Tris-HCl, pH 8.0—8.5. Water can
also be used but yield may be slightly lower.
d. Before use, add 150 µl, or 750 µl of sterilized water to the tube containing
2 mg, or 10 mg of proteinase K, respectively. For long term storage,
proteinase K solution should be kept at -20ºC.
Principle
This kit is designed for rapid isolation of genomic DNA from cells and bacteria.
The kit contains a membrane embedded column for binding up to 10 µg of
genomic DNA. Proteins, salts, and other impurities are washed away. Purified
genomic DNA can be used in most molecular biology experiments including
restriction enzyme digestion, PCR, Southern-blotting etc.
Procedures for Isolation of Genomic DNA from Cells or Bacteria
1. Sample Preparation.
A. Cell Cultures
(1) Cells grown in suspension
B. Bacteria Collection
Spin appropriate number of bacteria (about 106~107) at 6,000 x g (8,000 rpm) for
5 minutes at room temperature. Remove supernatant completely and resuspend
cells in 200 µl cold TE Buffer (not provided with kit), proceed to Step 2.
C. Paraffin Tissue
(1) Excise 25~30 mg paraffin tissue with a clean, sharp scalpel. Transfer to a 1.5
ml Eppendorf tube.
(2) Add 1.2 ml xylene (not provided with kit, Xylene is used to remove paraffin) to
the tube, vortex for 3 minutes.
(3) Centrifuge at 10,000 x g (12,000 rpm) for 5 minutes at room temperature.
(4) Discard the supernatant and keep the pellet.
(5) Add 1.2 ml of 100% ethanol to the tube. Gently vortex for 1 minute. Incubate
at room temperature for 1 minute.
(6) Centrifuge at 10,000 x g (12,000 rpm) for 5 minutes at room temperature.
Discard supernatant.
(7) Repeat step 5 to 6.
(8) Incubate at 37 ºC for 10-15 minutes to remove residual ethanol.

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 EZ-10 Genomic DNA Kit Handbook

(9) Resuspend the sample in 200 µl TE buffer, and proceed to Step 2.

2. Add 400 µl of Digestion Solution to 200 µl sample from step 1. Mix well. Add 3
µl of Proteinase K solution (2 mg/150 µl) to sample and incubate at 55 ºC for 5 Troubleshooting Guide
minutes.
1. Low yield
Do not add proteinase K solution directly to Digestion Solution.
a. Improper storage of starting material
Incubation period depends on the nature of sample. For cell cultures, 5
>>>Prepare fresh samples and use immediately.
minutes is generally enough to obtain complete lysate. For tissue samples
b. Too much or too less starting material
it requires 1-5 hours. Longer period incubation even overnight will not
>>>Reduce or increase starting material accordingly.
affect the result.
c. Incorrect preparation of buffers
If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
>>>Each step has to be strictly followed.
provided with kit), mix by vortexing, and incubate for 5 min at room
temperature before continuing with step 3. 2. RNA contamination
Perform optional RNase treatment according to the protocol.
3. Add 260 µl of 100% ethanol, and mix well. Apply the mixture onto an EZ-10
spin column that is placed in a 2.0 ml Collection Tube. Spin at 8,000 x g 3. OD260nm/OD280nm ratio outside 1.7-1.9 range
(10,000 rpm) for 2 minutes.
If the ratio of OD260nm/OD280nm is greater than 1.9, there may be traces of RNA
4. Discard the flow-through in the collection tube. Add 500 µl of Wash Solution, contamination. If the ratio of OD260nm/OD280nm is smaller than 1.7, there
and spin at 8,000 x g (10,000 rpm) for 2 minutes. may be protein contamination. Make sure the sample is mixed well after
proteinase K digestion.
5. Repeat Step 4.

6. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute 4. DNA does not perform well
to remove residual amount of Wash Solution. a. DNA Shearing
7. Place the EZ-10 column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl >>>Avoid repeated freezing and thawing of starting material; if samples are
Elution Buffer into the center part of membrane in the column. Incubate at RT too old, start with a new sample.
for 2 or 3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may b. Ethanol Carryover
increase recovery yield. >>>Spin additional steps before elution

8. Spin at 8,000 x g (10,000 rpm) for 2 minute to elute DNA from the column.

9. For long term storage, keep aliquots of purified genomic DNA at -20 ºC.
10. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of
50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.

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 EZ-10 Genomic DNA Kit Handbook

EZ-10 Spin Column Genomic DNA Minipreps Kit, Plant Procedures for Isolation of Genomic DNA from Plants
Kit Contents 1. Plant Tissue Sample Preparation: Grind plant tissue under liquid nitrogen to a
fine powder using a mortar and pestle. Transfer the powder immediately after
BS425, BS626,
Component liquid nitrogen to be evaporated to 1.5 ml Eppendorf tube. Do not allow the
50 Preps 250 Preps sample to thaw. Proceed immediately to Step 2.
RNase A (10 mg/ml) (a) 150 µl 750 µl
If following steps could not be performed immediately, it is recommended
PCL Solution 15 ml 75 ml to pause here and store the powder at -20 ºC.
PP Solution 2 ml 10 ml Avoid repeated freezing and thawing of stored samples, since this leads
to reduced DNA size and yield.
PB Solution 20 m 100 ml Incubation period depends on the nature of sample. Longer period
Wash Solution (b) 12 ml 2 x 30 ml incubation even overnight will not affect the result.
(c)
Better results could be achieved if starting material is less than 60 mg.
Elution Buffer 5 ml 25 ml The amount of samples should not exceed 100 mg per EZ-10 Spin
EZ-10 Column (with 2.0-ml Collection Tube) 50 250 Column.
Protocol 1 1 2. Check the volume of grounded material, and add equal volume
(approximately 150 µl) of PCL Solution (Plant Cell Lysis Solution).
Notes:
a. PCL Solution DOES NOT contain RNase A (100 µg/ml). Please add entire 3. Vortex and shake the tube several times. Incubate at 65ºC for 20 minutes,
contents of RNAse A into PCL Solution and store at 4ºC. PCL Solution vortex or pipette up and down to further remove any clumps. Clumped tissue
may form precipitates upon storage. If necessary, dissolve the precipitate will not lyse properly and will result in a lower yield of DNA.
by warming up to room temperature.2 mg, or 10 mg of proteinase K,
respectively. For long term storage, proteinase K solution should be kept If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
at -20ºC. provided with kit), mix by vortexing, and incubate for 5 min at room
temperature before continuing with step 4.
b. Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS425,
or 120 ml of 100% ethanol to 30 ml Wash Solution for BS626. For other 4. Add 25 µl PP Solution. Mix well. Keep the solution on ice for 15 minutes.
volumes of wash solution, simply add enough ethanol to make a 4:1 ratio
(volume of added ethanol:volume of Wash Solution = 4:1). 5. Centrifuge at 4 ºC, 8,000 x g (10,000 rpm) for 5 minutes. Apply the clear
lysate to an EZ-10 Spin Column.
c. Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or
water can be used, yield may be 20% lower.
6. Add 300 µl PB buffer to the EZ-10 Spin Column. Mix gently by inverting the
tube. Incubate the mixture for 3 minutes at room temperature. During
incubation, mix occasionally by inverting the tube.

7. Centrifuge at 4 ºC, 8,000 x g (10,000 rpm) for 2 minutes.

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 EZ-10 Genomic DNA Kit Handbook

8. Discard the flow-through in the Collection Tube. Add 500 µl of Wash Solution, EZ-10 Spin Column Genomic DNA Minipreps Kit, Animal
and spin at 8,000 x g (10,000 rpm) for 2 minutes. Kit Contents
9. Repeat Step 8. BS427, BS628,
Component
10. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute 50 Preps 250 Preps
to remove residual amount of Wash Solution. ACL Solution (a) 20 ml 100 ml
11. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl Elution PBS Solution 75 ml 2 x 200 ml
Buffer into the center part of membrane in the column. Incubate at RT for 2 or
3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may increase AB Solution 20 ml 100 ml
recovery yield. (b)
Proteinase K 20 mg 100 mg
12. Spin at 8,000 x g (10,000 rpm) for 2 minutes to elute DNA from the column. (c)
Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent Wash Solution 12 ml 2 x 30 ml
of 50 µg). Elution Buffer
(d)
5 ml 25 ml
13. For long term storage, keep aliquots of purified genomic DNA at -20ºC. EZ-10 Column (with 2.0-ml Collection Tube) 50 250

14. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent Protocol 1 1
of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.
Notes:
Troubleshooting Guide a. ACL Solution may form precipitates upon storage. If necessary, dissolve
1. Low yield the precipitate by warming the solution to 37ºC.

There are a number of variables that can cause low yield. b. Before use, add 1 ml or 5 ml of sterilized water to the tube containing 20
a. Inefficient homogenization. mg or 100 mg of Proteinase K. Keep solution at -20ºC.
>>>Ensure that the material is completely disrupted. If necessary, increase
time of homogenization. c. Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS427
b. Low DNA content of plant tissue. or 120 ml of 100% ethanol to 30 ml Wash Solution for BS628. For other
>>>Increase the amount of starting material to 200 mg. volumes of wash solution, simply add enough ethanol to make a 4:1 ratio
(volume of added ethanol:volume of Wash Solution = 4:1).
2. RNA contamination
RNase activity is weakened or lost. Add 30% additional RNAse A, and store d.Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or
solution at 4 ºC. water can be used, yield is generally 10% lower.

3. OD260nm/OD280nm ratio outside 1.7-1.9 range

If the ratio of OD260nm/OD280nm is greater than 1.9, there may be traces of

RNA contamination. If the ratio of OD260nm/OD280nm is smaller than 1.7,
there is a chance of protein contamination. Make sure the sample is mixed
well after PCL Solution.

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Procedures for Isolation of Genomic DNA from Animal
For Animal Tissue
1. Cut up to 30 mg of tissue and place in a 1.5 ml centrifuge tube.
2. Add 300 µl of ACL Solution (Animal Cell Lysis Solution) to 1.5 ml centrifuge
tubes and 20 µl of Proteinase K.
3. Incubate at 55ºC until tissues are completely lysed (usually 1-3 hours). Vortex
occasionally. Incubate in shaking water bath can reduce lysis time.
If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
provided with kit), mix by vortexing, and incubate for 5 min at room
temperature before continuing with step 4.
4. Cool to room temperature. Vortex for 20 seconds and centrifuge 10,000 x g
(12,000 rpm) for 5 minutes.
5. Pipette 300 µl of supernatant to a new Eppendorf tube, add 300 µl of AB
Solution. Mix by occasionally inverting tube, and keep for 2 minutes. Then
load all the solution to a EZ-10 Spin Column.
6. Centrifuge at 2,000 x g (4,000 rpm) for 2 minutes and discard the
flow-through.
7. Add 500 µl of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 2
minutes.
8. Repeat Step 7.
9. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute
to remove residual amount of Wash Solution.
10. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl Elution
Buffer into the center part of membrane in the column. Incubate at RT for 2 or
3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may increase
recovery yield.
11. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column.
12. For long term storage, keep aliquots of purified genomic DNA at -20 ºC.
13 Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent
of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarose gel.
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EZ-10 Genomic DNA Kit Handbook
For Rodent Tail
1. Place numbered 1.5 ml centrifuge tubes on dry ice.
2. Cut 0.5 cm to 1 cm from ends of tails and place in tubes.
3. Add 300 µl of ACL Solution to 1.5 ml centrifuge tubes and then add 20 µl of
Proteinase K.
4. Incubate at 55 ºC overnight with rocking; or for several hours with occasional
mild vortexing every 15 minutes.
If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
provided with kit), mix by vortexing, and incubate for 5 min at room
temperature before continuing with step 5.
5. Cool to room temperature. Vortex for 20 seconds and centrifuge at 10,000 x
g (12,000 rpm) for 5 minutes.
6. Pipette 300 µl of supernatant into to a new Eppendorf tube, add 300 µl of AB
Solution. Mix by occasionally inverting tube, and keep for 2 minutes. Then
load all the solution to a EZ-10 Spin Column.
7. Centrifuge 2,000 x g (4,000 rpm) for 2 minutes and discard the flow-through.
8. Add 500 µl of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 1 minute.
9. Repeat Step 8
10. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute
to remove residual amount of Wash Solution.
11. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl Elution
Buffer into the center part of membrane in the column. Incubate at RT for 2 or
3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may increase
recovery yield.
12. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column.
13 For long term storage, keep aliquots of purified genomic DNA at -20 ºC.
14 Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent
of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.
12

For Cultured Animal Cell
1. Centrifuge the appropriate number of cells (>5x106) for 5 minutes at 200 x g
(1,200 rpm).
2. Resuspend pellet in 500 µl of PBS Solution.
3. Wash the cells 2 times with PBS Solution.
4. Resuspend pellet in 300 µl of ACL solution buffer.
5. Add 20 µl of Proteinase K, mix well and Incubate at 55 ºC for 10 minutes.
If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
provided with kit), mix by vortexing, and incubate for 5 min at room
temperature before continuing with step 6.
6. Cool to room temperature. Vortex for 20 seconds and centrifuge 10,000 x g
(12,000 rpm) for 5 minutes.
7. Pipette 200 µl of supernatant to a new Eppendorf tube, add 200 µl of AB
Solution. Mix by occasionally inverting tube, and keep for 2 minutes. Then
load all the solution to a EZ-10 Spin Column.
8. Centrifuge at 2,000 x g (4,000 rpm) for 2 minutes and discard the
flow-through.
9. Add 500 µl of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 1 minute.
10. Repeat Step 9.
11. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute
to remove residual amount of Wash Solution.
12. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl Elution
Buffer into the center part of membrane in the column. Incubate at RT for 2 or
3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may increase
recovery yield.
13 Spin at 8,000 x g (10,000 rpm) for 2 minutes to elute DNA from the column.
14 For long term storage, keep aliquots of purified genomic DNA at -20 ºC.
15 Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent
of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.
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EZ-10 Genomic DNA Kit Handbook
From Paraffin Tissue
1. Excise 25~30 mg paraffin tissue with a clean, sharp scalpel and transfer to a
1.5 ml Eppendorf tube.
2. Add 1.2 ml xylene (not included in the kit) to the tube, then vortex for 3
minutes. Xylene is used to remove paraffin.
3. Centrifuge at 10,000 x g (12,000 rpm) for 5 minute at room temperature.
4. Discard the supernatant and keep the pellet.
5. Add 1.2 ml 100% of ethanol to the tube. Gently vortex for 1 minute. Incubate
at room temperature for 1 minute.
6. Centrifuge at 10,000 x g (12,000 rpm) for 5 minute at room temperature.
Discard supernatant.
7. Repeat step 4 to 6.
8. Incubate at 37 ºC for 10-15 minutes to remove residual ethanol.
9. Resuspend the sample in 200 µl TE buffer, and proceed immediately to Step
10.
10. Add 300 µl of ACL Solution (Animal Cell Lysis Solution) and then add 20 µl of
Proteinase K.
If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
provided with kit), mix by vortexing, and incubate for 5 min at room
temperature before continuing with step 11.
11. Incubate at 55 ºC until the tissue is completely lysed (usually 1-3 hours).
Vortex occasionally. Incubation in shaking water bath can reduce lysis time.
12. Cool to room temperature. Vortex for 20 seconds and centrifuge at 10,000 x
g (12,000 rpm) for 5 minutes.
13 Pipette 300 µl of supernatant to a new Eppendorf tube, add 300 µl of AB
Solution. Mix by occasionally inverting tube, and keep for 2 minutes. Then
load all the solution to a EZ-10 Spin Column.
14 Centrifuge at 2,000 x g (4,000 rpm) for 2 minutes and discard the
flow-through.
15 Add 500 µl of Wash Solution, and spin at 6,000 x g (8,000 rpm) for 1 minute.
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 EZ-10 Genomic DNA Kit Handbook

16. Repeat Step 15. EZ-10 Spin Column Genomic DNA Minipreps Kit, Blood
17. Discard the flow-through and spin at 8,000 x g (10,000 rpm) for an additional Kit Contents
minute to remove residual amount of Wash Solution.
BS483, BS684
Component
18. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl Elution 50 Preps 250 Preps
Buffer into the center part of membrane in the column. Incubate at RT for 2 or
3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may increase TBP Buffer 120 ml 5 x 120 ml
recovery yield. (a)
TBM Buffer 25 ml 125 ml
19. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column TE (pH 8.0) 15 ml 75 ml
(b)
20. For long term storage, keep aliquots of purified genomic DNA at -20 ºC. Proteinase K 2 mg 10 mg
(c)
Wash Solution 12 ml 2 x 30 ml
21. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent
of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel. Elution Buffer (d)
5 ml 25 ml

EZ-10 Column (with 2.0-ml Collection Tube) 50 250

Protocol 1 1

Notes:
a. TBM Buffer may form a precipitate upon storage. If necessary, dissolve the
precipitate by warming at 37 ºC.
b. Before use, add 150 µl or 750 µl of sterilized water to the tube containing 2
mg or 10 mg of Proteinase K respectively. Keep at -20 ºC for long term
storage.
c. Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS483,
or 120ml of 100% ethanol to 30ml Wash Solution for BS684. For other
volumes of wash solution, simply add enough ethanol to make a 4:1 ratio
(volume of added ethanol:volume of Wash Solution = 4:1).

d. Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or

water can be used, yield is generally 20% lower.
Storage of Blood
Whole blood samples treated with EDTA, ACD or heparin can be used, and
may be either fresh or frozen. For short term storage (up to 10 days), collect blood
in tubes containing EDTA as an anticoagulant, and store tubes at 2-8 ºC. It is
recommended to store blood sample less than 3 days as DNA degradation may
occur. For long term storage, collect blood in tubes containing a standard
anticoagulant (preferably EDTA if high molecular weight DNA is required) and
store at -80 ºC.

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 EZ-10 Genomic DNA Kit Handbook

Blood Collection and Treatment 12. For long term storage, keep aliquots of purified genomic DNA at -20 ºC.
For every 6 ml of whole blood sample, add 1 ml of anticoagulant (0.5M EDTA
pH 8.0, or ACD, 0.48% Citric Acid, 1.32% Sodium Citrate, 1.47% Glucose). 13. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of
50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.
Procedures for Extraction Genomic DNA from Blood
1. Harvest 0.5 ml of whole blood in 2.0 ml centrifuge tube. Add 0.8 ml TBP Buffer Troubleshooting Guide
to the tube. Vortex gently and let the tube stand for 1 min at room
1. Low yield
temperature. The erythrocytes should be lysed and solution should appear
clear red. There are a number of variables that can cause low yield.
a. Each step has to be strictly followed.
2. Spin at 2,000 x g (4,000 rpm) for 3 minutes. Discard supernatant. b. Make sure column binding capacity 10 µg is not exceeded.
3. Repeat Step 2. If the supernatant and blood pellet remain red in color, repeat 2. RNA contamination
step 2. If the blood pellet looks mauve or colorless, dissolve the pellet in 200
µl TE buffer and continue with step 4. RNase activity is weakened or lost. Add 30% additional RNAse A, and store
solution at 4 ºC.
4. Add 0.5 ml TBM Buffer to the centrifuge tube. Vortex the tube vigorously and
then add 3 µl Proteinase K. Incubate at 55 ºC for 30 minutes. 3. Sample floats upon loading in agarose gel
The sample contains ethanol from washing step. Discard the liquid waste
If RNA-free genomic DNA is required, add 20 µl RNase A (10 mg/ml, not
from the collection tube after washing step, and spin again for additional two
provided with kit), mix by vortexing, and incubate for 5 min at room minutes. Before elution step, incubate the column at 50 ºC for ~5 min and
temperature before continuing with step 4. allow ethanol to evaporate completely.
5. If insoluble material is visible, centrifuge for 2 minutes at 2,500 x g (5,000
rpm). Transfer supernatant to another 2.0 ml centrifuge tube and add 260 µl
100% ethanol. PRODUCTS ARE INTENDED FOR BASIC SCIENTIFIC
RESEARCH ONLY!
6. Apply the mixture to EZ-10 column that is in a 2.0 ml Collection Tube. Spin at
8,000 x g (10,000 rpm) for 2 minutes. Discard the flow-through in the NOT INTENDED FOR HUMAN OR ANIMAL USE!
collection tube.
7. Add 500 µl of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 1 minute.
8. Repeat Step 7.
9. Discard the flow-through. Spin at 8,000 x g (10,000 rpm) for an additional
minute to remove any residual amount of Wash Solution.
10. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 µl Elution
Buffer into the center part of membrane in the column. Incubate at RT for 2 or
3 minutes. Incubating the tube at 37ºC or 50ºC for 2 minutes may increase
recovery yield.
11. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column.

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