Agilent Technologies PCR Polishing INSTRUCTION MANUAL

PCR Polishing Kit

INSTRUCTION MANUAL

Catalog #200409

Revision A

For In Vitro Use Only

200409-12

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PCR Polishing Kit

CONTENTS

Materials Provided.............................................................................................................................. 1

Storage Conditions.............................................................................................................................. 1

Additional Materials Required .......................................................................................................... 1

Introduction......................................................................................................................................... 2

Precautionary Notes............................................................................................................................ 2

Protocol ................................................................................................................................................ 3

PCR Polishing Reaction ........................................................................................................ 3

Standard Blunt-End Ligation of Control DNA...................................................................... 5

Preparation of Media and Reagents.................................................................................................. 6

References ............................................................................................................................................ 6

Endnotes............................................................................................................................................... 6

MSDS Information.............................................................................................................................. 6

PCR Polishing Kit

ATERIALS PROVIDED

M

Materials provideda Quantity

Cloned Pfu DNA polymerase

10x cloned Pfu DNA polymerase buffer

Control DNA (pUC19)f 50 μl (10 ng/μl)

10 mM dNTP mix

a

The PCR Polishing Kit contains enough reagents to perform 40 reactions.

b

Stratagene Catalog #600153 (100 U), #600154 (500 U), #600159 (1000 U), and #600160 (5000 U).

c

Do not store the cloned Pfu DNA polymerase or the 10 mM dNTP mix in a frost-free freezer.

d

We recommend aliquoting the 10x cloned Pfu DNA polymerase buffer and 10 mM dNTP mix into smaller volumes
following initial thawing. Avoid multiple freeze–thaw cycles to achieve maximum levels of incorporation.

e

See Preparation of Media and Reagents.

f

The control DNA is linearized with 3´-end extensions (pUC19, Hind II-digested and Taq DNA polymerase treated).

c,d

50 μl (2.5 mM each)

b,c,

100 U (2.5 U/μl)

d,e

1.0 ml

STORAGE CONDITIONS

Cloned Pfu DNA Polymerase: –20°C
10× Cloned Pfu DNA Polymerase Buffer: –20°C
Control DNA: –20°C
10 mM dNTP mix: –20°C (for up to 3 months); –80°C (for long-term storage)

ADDITIONAL MATERIALS REQUIRED

Sterile microcentrifuge tubes (0.5 ml)
Mineral oil
Sterile distilled water (dH

Revision A © Agilent Technologies, Inc. 2009.

O)

2

PCR Polishing Kit 1

INTRODUCTION

The PCR Polishing Kit is designed to increase the blunt-ended cloning
efficiencies associated with polymerase chain reaction (PCR)-generated
fragments.
polymerases (e.g., T7, modified T7, Taq, Vent
exhibit terminal deoxynucleotidyltransferase-like activity (extendase).

1,2

Recent studies have shown that many species of DNA

®

and Klenow fragment)

R

3,4

It
has been found that the 3´-end nucleotide extension of PCR products by
DNA polymerases is both nucleotide and polymerase specific. Each DNA
polymerase, including the Klenow fragment, has characteristic terminal
extendase activity with no consistent pattern of 3´-end modification.
Therefore, it cannot be assumed that all DNA polymerases create blunt-

,

ended fragments. Fortunately, Pfu DNA polymerase*

does not exhibit any

DNA extendase and can be used to create blunt-ended fragments following

5–7

PCR.

Due to the unique 3´ → 5´ exonuclease (proofreading) activity of
the Pfu DNA polymerase, Taq DNA polymerase-generated PCR products
can be polished with Pfu DNA polymerase following temperature cycling to
create blunt-ended DNA fragments for use in cloning, mutagenesis and
cDNA construction.

The PCR Polishing Kit is designed to polish the ends of the 3´-overhang
extensions of polymerase-generated DNA fragments directly from a PCR
amplification reaction. The PCR Polishing Kit can also be used to perform
complete fill-in of 5´ overhangs to generate blunt ends. Dramatic increase in
the population of blunt-ended DNA fragments following polishing treatment
results in a drastic increase in overall experimental efficiency associated
with procedures utilizing blunt-ended ligation.

PRECAUTIONARY NOTES

♦ Before PCR polishing, it may be advantageous to verify the PCR

products by agarose gel analysis in order to estimate the approximate
concentration of PCR products and to ensure that the correct PCR
products have been created following thermal amplification. PCR
polishing is conducted using an aliquot of the PCR amplification
reaction and will therefore polish the ends of all DNA fragments

present. In a typical 100-μl PCR amplification reaction that has been
cycled ~30 rounds exhibiting a strong band following agarose gel

analysis, 5 μl of product can be used for PCR polishing.

♦ Before performing a 5´-fill-in reaction, the sample DNA should be

extracted with phenol–chloroform and ethanol precipitated
(see Optional Purification Protocols).

* U.S. Patent Nos. 6,489,150, 5,948,663, 5,866,395 and 5,545,552 and patents pending.

2 PCR Polishing Kit

PROTOCOL

PCR Polishing Reaction

For polishing PCR-generated DNA fragments, transfer an aliquot of PCR
product directly from the reaction tube into a sterile 0.5-ml microcentrifuge
tube. For 5´-fill-in reactions, aliquot purified DNA (10–500 ng) into a sterile

0.5-ml microcentrifuge tube.

1. Add the following components in order to the sterile 0.5-ml

microcentrifuge tubes:

Control Reaction

5.0 μl (50 ng) of control DNA

1.0 μl of 10× cloned Pfu DNA
polymerase buffer

1.0 μl of dNTP mix

1.0 μl of cloned Pfu DNA
polymerase (2.5 U/μl)

dH

O to a total of 10 μl

2

Gently mix the components of both microcentrifuge tubes and add a

mineral oil overlay.

2. Incubate the control and sample reactions at 72°C for 30 minutes. After
the 30-minute incubation, remove the reactions to ice.

3. End-polished DNA fragments may be added directly to ligation
reactions.

Note Pfu DNA polymerase has very little activity when used in

ligation reactions (25°C). Therefore, PCR-polished DNA
fragments need not be further purified for use in ligation
reactions. However, an optional purification protocol has
been outlined in the following section (see Optional
Purification Protocols).

Sample Reaction

5.0 μl (10–500 ng) of PCR DNA

1.0 μl of 10× cloned Pfu DNA
polymerase buffer

1.0 μl of dNTP mix

1.0 μl of cloned Pfu DNA
polymerase (2.5 U/μl)

dH

O to a total of 10 μl

2

PCR Polishing Kit 3

Optional Purification Protocols

The PCR-polished DNA fragments may be further purified to remove the
Pfu DNA polymerase and excess dNTPs or for buffer exchange. The
StrataPrep PCR purification kit [Stratagene Catalog #400771 (50 preps) and
#400773 (250 preps)] can be used in the place of phenol–chloroform
extraction procedures. Conventional phenol–chloroform extraction is
outlined below:

1. Aliquot the PCR-polished reaction mixture, avoiding the mineral oil
overlay, to a sterile 0.5-ml microcentrifuge tube. Adjust the final

volume to 100 μl with TE buffer.

2. Add an equal volume of phenol, vortex and remove the top aqueous
phase to a sterile 0.5-ml microcentrifuge tube.

3. Add an equal volume of chloroform, vortex and remove the top
aqueous phase to a sterile 0.5-ml microcentrifuge tube.

4. Precipitate the DNA using ammonium acetate as follows:

§

a. Add 1/10 volume of 10× STE buffer.

§

b. Add an equal volume of 4 M ammonium acetate to the sample.

c. Add 2.5 volumes of room temperature 100% (v/v) ethanol.

d. Immediately spin in a microcentrifuge at room temperature for

20 minutes at 10,000 × g to pellet the DNA.

e. Carefully remove and discard the supernatant.

f. Wash the DNA pellet with 200 μl of 70% (v/v) ethanol.

g. Spin in a microcentrifuge at room temperature for 10 minutes at

10,000 × g. Carefully remove the ethanol with a pipet.

h. Dry the DNA pellet under vacuum.

i. Dilute the DNA into TE buffer or into a preferred diluent prior to

ligation.

§

See Preparation of Media and Reagents.

4 PCR Polishing Kit

Standard Blunt-End Ligation of Control DNA

The unpolished control DNA (pUC19) contains 3´-end nucleotide
extensions and will ligate at an extremely reduced efficiency in the presence
of T4 DNA ligase. PCR polishing removes 3´-end nucleotide extensions
created by DNA polymerases and will produce blunt-ended DNA molecules
which will religate at high efficiency in the presence of T4 DNA ligase. For
the efficient cloning of blunt-ended PCR products, we recommend using the
PCR-Script Amp cloning kit (Stratagene Catalog 211190).
of rATP and T4 DNA ligase, ligations of control DNA can be performed as
follows:

8,9

In the presence

Unpolished Control DNA

1.0 μl (10 ng) of unpolished
control DNA (pUC19)

1.0 μl of 10× cloned Pfu DNA
polymerase buffer

0.5 μl of rATP (10 mM stock)

1.0 μl of T4 DNA ligase
(4 Weiss U)

6.5 μl of dH

O

2

PCR-Polished Control DNA

2.0 μl (10 ng) of PCR-polished
control DNA (pUC19)

1.0 μl of 10× cloned Pfu DNA
polymerase buffer

0.5 μl of rATP (10 mM stock)

1.0 μl of T4 DNA ligase
(4 Weiss U)

5.5 μl of dH2O

1. Incubate the ligations at room temperature (22°C) for 1 hour.

2. Transform 1–2 μl of the ligated DNA into competent E. coli cells. The
control DNA (pUC19) provided is an ampicillin-resistance encoding
plasmid and should be spread onto LB–ampicillin agar plates
(see Preparation of Media and Reagents) following transformation.

3. Following the overnight incubation at 37°C, the transformation plates
containing PCR-polished control DNA should exhibit a >20-fold
increase in colony forming units (cfu) when compared to unpolished
control DNA transformants.

PCR Polishing Kit 5

PREPARATION OF MEDIA AND REAGENTS

10× Cloned Pfu DNA Polymerase
Buffer

200 mM Tris-HCl (pH 8.75)
100 mM KCl
100 mM (NH4)2SO4
20 mM MgSO

4

1 mg/ml of bovine serum albumin (BSA)

®

1% Triton

X-100

LB Agar (per Liter)

10 g of NaCl
10 g of tryptone
5 g of yeast extract
20 g of agar
Add deionized H

O to a final volume of

2

1 liter
Adjust pH to 7.0 with 5 N NaOH
Autoclave
Pour into petri dishes

(~25 ml/100-mm plate)

REFERENCES

10× STE Buffer

1 M NaCl
200 mM Tris-HCl (pH 7.5)
100 mM EDTA

TE Buffer

10 mM Tris-HCl (pH 7.5)
1 mM EDTA

LB–Ampicillin Agar (per Liter)

1 liter of LB agar, autoclaved
Cool to 55°C
Add 10 ml of 10-mg/ml filter-sterilized

ampicillin

Pour into petri dishes

(~25 ml/100-mm plate)

1. Costa, G. L. and Weiner, M. P. (1994) Strategies 7(1):8.

2. Costa, G. L. and Weiner, M. P. (1994) Strategies 7(2):47-48.

3. Clark, J. M. (1988) Nucleic Acids Res 16(20):9677-86.

4. Hu, G. (1993) DNA Cell Biol 12(8):763-70.

5. Costa, G. L. and Weiner, M. P. (1994) PCR Methods Appl 3(5):S95-106.

6. Costa, G. L., Grafsky, A. and Weiner, M. P. (1994) PCR Methods Appl 3(6):338-45.

7. Costa, G. L. and Weiner, M. P. (1994) Nucleic Acids Res 22(12):2423.

8. Bauer, J. C., Deely, D., Braman, J., Viola, J. and Weiner, M. P. (1992) Strategies
5(3):62-64.

9. Costa, G. L., Sanchez, T. and Weiner, M. P. (1994) Strategies 7(2):52.

ENDNOTES

Vent
Triton

®

is a registered trademark of New England Biolabs, Inc.

R

®

is a registered trademark of Rohm and Haas Co.

MSDS INFORMATION

The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at
http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated MSDS’s
in a print-ready format. MSDS documents are not included with product shipments.
.

6 PCR Polishing Kit