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PCR Purification

Gives an easy protocol for DNA purification after pcr or endonuclease digestion

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theopanas2000
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© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
32 views

PCR Purification

Gives an easy protocol for DNA purification after pcr or endonuclease digestion

Uploaded by

theopanas2000
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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QUICK REFERENCE

PureLink® PCR Purification Kit


Catalog numbers K3100-01 and K3100-02
Publication Part Number 7015021 MAN0004375 Revision Date 14 September 2011

Purifying PCR Products


Prepare Buffers
Buffer Used for Cat. no. Cat. no.
K3100-01 K3100-02
Binding Buffer (B2) purifying 100 bp to 12 kb dsDNA Add 10 mL Add 48 mL
PCR fragments isopropanol isopropanol
Binding Buffer HC (B3) removing primer dimers or short Add 2.3 mL Add 11 mL
spurious PCR products (<300 bp) isopropanol isopropanol
Wash Buffer (W1) all procedures Add 64 mL Add 320 mL
ethanol ethanol

Procedure for Purifying PCR Products


1. Combine. Add 4 volumes of Binding Buffer B2 or B3 with isopropanol
(see preceding table) to 1 volume of a PCR sample (50–100 μL). Mix well.

2. Load. Pipet the sample into a PureLink® Spin Column in a Collection Tube.
Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.

3. Wash. Re-insert the column into the Collection Tube and add 650 µL Wash
Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute.
Discard the flow-through and place the column in the same Collection Tube.
Centrifuge the column at maximum speed for 2–3 minutes.

4. Elute. Place the column into a clean 1.7-mL Elution Tube (supplied with
the kit). Add 50 µL Elution Buffer to the center of the column. Incubate
the column at room temperature for 1 minute. Centrifuge the column at
maximum speed for 2 minutes. The elution tube contains the purified PCR
product. Store the purified DNA at 4°C for immediate use or at −20°C for
long-term storage.
Intended Use
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Troubleshooting
Problem Solution
Low DNA • Check the amplicon on a gel to verify the PCR product prior to
yield purification.
• Always mix 1 volume of PCR (50–100 μL) with 4 volumes of Binding
Buffer (B2 or B3).
• Be sure to add 100% isopropanol to the Binding Buffers (B2 and B3).
• Be sure to add 96–100% ethanol to Wash Buffer (W1).
• Add Elution Buffer to the center of the column. Incubate the tube with
Elution Buffer for 1 minute before centrifugation.
Primer To efficiently remove primer dimers or short, spurious PCR products
dimers are (<300 bp), use Binding Buffer B3. Binding Buffer B3 is specifically
present designed to remove <300 bp DNA fragments, eliminating the need for gel
purification.
Enzymatic To remove Wash Buffer, discard Wash Buffer flow-through from the Wash
reactions Tube. Place the column into the Wash Tube and centrifuge the column at
are >12,000 × g for 2–3 minutes to completely dry the column.
inhibited

Limited Use Label License: Research Use Only


The purchase of this product conveys to the purchaser the limited, non-transferable right
to use the purchased amount of the product only to perform internal research for the
sole benefit of the purchaser. No right to resell this product or any of its components is
conveyed expressly, by implication, or by estoppel. This product is for internal research
purposes only and is not for use in commercial applications of any kind, including,
without limitation, quality control and commercial services such as reporting the results
of purchaser’s activities for a fee or other form of consideration. For information on
obtaining additional rights, please contact [email protected] or Out Licensing, Life
Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

©2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned
herein are the property of Life Technologies Corporation or their respective owners.

Headquarters
5791 Van Allen Way | Carlsbad, CA 92008 USA
Phone +1 760 603 7200 | Toll Free in USA 800 955 6288
For support visit www.invitrogen.com/support
email [email protected]
www.lifetechnologies.com

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