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Wizard SV Genomic DNA Purification System Quick Protocol PDF

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Pramusita Yoga Daniswara
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486 views4 pages

Wizard SV Genomic DNA Purification System Quick Protocol PDF

Uploaded by

Pramusita Yoga Daniswara
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 4

Printed in USA.

Revised 2/12
Part #9FB069
P R O T O C O L
Quick
3
6
4
0
M
A
0
2
_
2
A
Add Wizard


SV Lysis Buffer to
each sample. Vortex.
Transfer warm
lysate to
minicolumn.
Apply vacuum
until lysate passes
through column.
Wash.
Dry matrix
with vacuum.
Transfer spin
column to
microcentrifuge
tube. Add
Nuclease-Free
Water and
centrifuge.
Digest mouse tail
clipping or tissue
sample in Proteinase K
Digestion Solution.
Incubate at 55C
for 1618 hours.

Purification of Genomic DNA from Mouse Tail Clippings or Animal Tissues Using Vacuum
Wizard

SV Genomic DNA Purification System


INSTRUCTIONS FOR USE OF PRODUCTS A2360, A2361 AND A2365.
Sample Preparation
1. Cut a 0.5 to 1.2cm length of mouse tail or weigh up to 20mg of tissue sample.
Cut the clipping or tissue sample into two pieces and place them in a 1.5ml
microcentrifuge tube.
2. Add 275l of Digestion Solution Master Mix to each tube.
3. Incubate the sample tubes overnight (1618 hours) in a 55C heat block.
4. Add 250l of Wizard

SV Lysis Buffer to each sample. Vortex.
5. Process the lysate as soon as possible after adding the Lysis Buffer. If frozen
at 70C, lysates must be thawed and heated at 55C for one hour prior to
processing. Lysates must be warm for processing.
Purification of Genomic DNA Using a Vac-Man

Vacuum Manifold
6. For each lysate, use one Wizard

SV Minicolumn. Attach one Miniprep


Vacuum Adapter with Luer-Lok

fitting to one port of the manifold. Gently


press a minicolumn into the vacuum adapter. Ensure that all unused ports of
the vacuum manifold are closed.
7. Transfer the prepared sample lysate to the Wizard

SV Minicolumn.
8. Apply a vacuum until the lysate passes through the minicolumn. After the
lysate has passed through the column, close the Luer-Lok

stopcock.
9. Add 800l of Column Wash Solution (CWA; with 95% ethanol added) to
the minicolumn. Apply a vacuum until the solution has passed through the
column, then close the port. Repeat this step for a total of 4 washes.
10. Open each port and continue to pull a vacuum for 4 minutes to dry the
binding matrix.
11. Close each port. Turn off the vacuum source and break the vacuum.
12. Remove the Wizard

SV Minicolumn and place in a new 1.5ml tube. Add


250l of room temperature Nuclease-Free Water to the minicolumn. Incubate
for 2 minutes at room temperature.
13. Place the minicolumn/tube assembly into the centrifuge and spin at
13,000 x g for 1 minute. Repeat Steps 1213 for a total of 2 elutions (500l).
14. Remove the minicolumn. Store the purified DNA at 20 to 70C.
See additional protocol information in Technical Bulletin #TB302, available online at:
www.promega.com/protocols
ORDERING/ TECHNICAL INFORMATION:
www.promega.com Phone 608-274-4330 or 800-356-9526 Fax 608-277-2601
20022003, 2009, 2012 Promega Corporation. All Rights Reserved.
Digestion Solution Volume
Master Mix per Sample
Nuclei Lysis Solution 200l
0.5M EDTA (pH 8.0) 50l
Proteinase K, 20mg/ml 20l
RNase A Solution, 4mg/ml 5l
Total Volume 275l
Printed in USA. Revised 2/12.
Part #9FB069
Purification of Genomic DNA from Tissue Culture Cells Using Vacuum
Wizard

SV Genomic DNA Purification System


INSTRUCTIONS FOR USE OF PRODUCTS A2360, A2361 AND A2365. P R O T O C O L
Quick
3
6
3
9
M
A
0
2
_
2
A
Add Wizard

SV
Lysis Buffer to
each sample.
Pipet to mix.
Transfer lysate
to minicolumn.
Apply vacuum
until lysate passes
through column.
Wash.
Dry matrix
with vacuum.
Transfer spin
column to
microcentrifuge
tube. Add
Nuclease-Free
Water and
centrifuge.

Sample Preparation
1. Use at least 1 10
4
cells to a maximum of 5 10
6
cells. Wash the cells once
with 1X PBS.
2. Add 150l of Wizard

SV Lysis Buffer to the washed cells in a tissue culture


plate. Mix lysate by pipetting.
3. If the cell lysates will not be used immediately, they can be frozen at 70C
until needed.
Purification of Genomic DNA Using a Vac-Man

Vacuum Manifold
4. For each lysate, use one Wizard

SV Minicolumn. Attach one Miniprep


Vacuum Adapter with Luer-Lok

fitting to one port of the manifold. Gently


press a minicolumn into the vacuum adapter. Ensure that all unused ports of
the vacuum manifold are closed.
5. Transfer the prepared sample lysate to the Wizard

SV Minicolumn.
6. Apply a vacuum until the lysate passes through the minicolumn. After the
lysate has passed through the column, close the Luer-Lok

stopcock.
7. Add 800l of Column Wash Solution (CWA; with 95% ethanol added) to
the minicolumn. Apply a vacuum until the solution has passed through the
column, then close the port. Repeat this step for a total of 4 washes.
8. Open each port and continue to pull a vacuum for 4 minutes to dry the
binding matrix.
9. Close each port. Turn off the vacuum source and break the vacuum.
10. Remove the Wizard

SV Minicolumn and place in a new 1.5ml tube. Add


250l of room temperature Nuclease-Free Water to the minicolumn. Incubate
for 2 minutes at room temperature.
11. Place the minicolumn/tube assembly into the centrifuge and spin at
13,000 x g for 1 minute. Total elution volume will be approximately 250l.
12. Remove the minicolumn. Store the purified DNA at 20 to 70C.
See additional protocol information in Technical Bulletin #TB302, available online at:
www.promega.com/protocols
ORDERING/ TECHNICAL INFORMATION:
www.promega.com Phone 608-274-4330 or 800-356-9526 Fax 608-277-2601
20022003, 2009, 2012 Promega Corporation. All Rights Reserved.
Printed in USA. Revised 2/12.
Part #9FB069
Purification of Genomic DNA from Mouse Tail Clippings or Animal Tissues Using a
Microcentrifuge
Wizard

SV Genomic DNA Purification System


INSTRUCTIONS FOR USE OF PRODUCTS A2360, A2361 AND A2365. P R O T O C O L
Quick

ORDERING/ TECHNICAL INFORMATION:


www.promega.com Phone 608-274-4330 or 800-356-9526 Fax 608-277-2601
3
6
4
1
M
A
0
2
_
2
A
Add Wizard

SV
Lysis Buffer to
each sample.
Transfer lysate
to a Wizard

SV
Minicolumn
Assembly.
Centrifuge to bind DNA.
Wash.
Transfer spin column
to a new 1.5ml tube.
Add Nuclease-Free
Water. Incubate at
room temperature.
Elute genomic DNA.
Digest mouse tail
clipping or tissue
sample in Proteinase K
Digestion Solution.
Incubate at 55C
for 1618 hours.
Centrifuge to dry
the binding matrix.
20022003, 2009, 2012 Promega Corporation. All Rights Reserved.
Sample Preparation
1. Cut a 0.5 to 1.2cm length of mouse tail or weigh up to 20mg of tissue sample.
Cut the clipping or tissue sample into two pieces and place them in a 1.5ml
microcentrifuge tube.
2. Add 275l of Digestion Solution Master Mix to each tube.
3. Incubate the sample tubes overnight (1618 hours) in a 55C heat block.
4. Add 250l of Wizard

SV Lysis Buffer to each sample. Vortex.
5. Process the lysate as soon as possible after adding the Lysis Buffer. If frozen
at 70C, lysates should be thawed and heated at 55C for one hour prior to
processing. Lysates must be warm for processing.
Purification of Genomic DNA from Lysate Using a Microcentrifuge
6. Transfer each sample lysate from the 1.5ml tube to a separate Wizard

SV
Minicolumn Assembly.
7. Spin the Assembly at 13,000 g for 3 minutes.
8. Remove minicolumn from the Assembly and discard the liquid in the
Collection Tube. Replace the minicolumn into the Collection Tube.
9. Add 650l of Column Wash Solution (CWA; with 95% ethanol added) to each
assembly. Centrifuge at 13,000 g for 1 minute. Discard the liquid from the
Collection Tube. Repeat this step for a total of 4 washes.
10. Discard the liquid from the Collection Tube and reassemble the minicolumn
assembly. Centrifuge for 2 minutes at 13,000 g to dry the binding matrix.
11. Transfer the Wizard

SV Minicolumn to a new 1.5ml tube. Add 250l of room


temperature Nuclease-Free Water. Incubate for 2 minutes at room temperature.
12. Centrifuge the minicolumn/elution tube assembly at 13,000 g for 1 minute.
Do not discard the liquid in the elution tube.
13. Add an additional 250l of Nuclease-Free Water and incubate at room
temperature for 2 minutes. Centrifuge the minicolumn/elution tube assembly
at 13,000 g for 2 minutes.
14. Remove the minicolumn and store the purified DNA at 20 to 70C.
See additional protocol information in Technical Bulletin #TB302, available online at:
www.promega.com/protocols
Digestion Solution Volume
Master Mix per Sample
Nuclei Lysis Solution 200l
0.5M EDTA (pH 8.0) 50l
Proteinase K, 20mg/ml 20l
RNase A Solution, 4mg/ml 5l
Total Volume 275l
Isolation of Genomic DNA from Tissue Culture Cells Using a Microcentrifuge
Wizard

SV Genomic DNA Purification System


INSTRUCTIONS FOR USE OF PRODUCTS A2360, A2361 AND A2365. P R O T O C O L
Quick
Add Wizard

SV
Lysis Buffer
to each sample.
Pipet to mix.
3
6
3
8
M
A
0
2
_
2
A
Transfer lysate to a
Wizard

SV
Minicolumn Assembly.
Centrifuge to bind DNA.
Wash.
Centrifuge to dry
the binding matrix.
Transfer spin
column to a new 1.5ml
tube. Add Nuclease-Free
Water. Incubate at room
temperature.
Centrifuge to elute DNA.

ORDERING/ TECHNICAL INFORMATION:


www.promega.com Phone 608-274-4330 or 800-356-9526 Fax 608-277-2601
Sample Preparation
1. Use at least 1 10
4
cells to a maximum of 5 10
6
cells. Wash the cells once
with 1X PBS.
2. Add 150l of Wizard

SV Lysis Buffer to the washed cells in a tissue culture


plate. Mix lysate by pipetting.
3. If the cell lysates will not be used immediately, they can be frozen at 70C
until needed.
Purification of Genomic DNA from Tissue Culture Cell Lysate Using a
Microcentrifuge
4. Transfer each sample lysate from the tissue culture plate to a separate Wizard

SV Minicolumn Assembly.
5. Spin the Assembly at 13,000 g for 3 minutes.
6. Remove minicolumn from the Assembly and discard the liquid in the
Collection Tube. Replace the minicolumn into the Collection Tube.
7. Add 650l of Column Wash Solution (CWA; with 95% ethanol added) to each
assembly. Centrifuge at 13,000 g for 1 minute. Discard the liquid from the
Collection Tube. Repeat this step for a total of 4 washes.
8. Discard the liquid from the Collection Tube and reassemble the minicolumn
assembly. Centrifuge for 2 minutes at 13,000 g to dry the binding matrix.
9. Transfer the Wizard

SV Minicolumn to a new 1.5ml tube. Add 250l of room


temperature Nuclease-Free Water. Incubate for 2 minutes at room temperature.
10. Centrifuge the minicolumn/elution tube assembly at 13,000 g for 1 minute.
Total elution volume will be 250l.
11. Remove the minicolumn and store the purified DNA at 20 to 70C.
See additional protocol information in Technical Bulletin #TB302, available online at:
www.promega.com/protocols
Printed in USA. Revised 2/12.
Part #9FB069
20022003, 2009, 2012 Promega Corporation. All Rights Reserved.

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