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LONG PCR CORE KIT
Product No. LCOR-1
Bulletin No. MB-415
October 1998
The Polymerase Chain Reaction (PCR)’ is a powerful
molecular biology technique now standardly used for
cloning, sequencing and genome mapping. The
primary enzyme used in PCR is Taq DNA polymerase.
Polymerase Chain Reaction using Taq DNA
polymerase is generally limited to amplifications up to
5 kb in part because Taq DNA polymerase has no
3’→5’ exonuclease or “proofreading” activity, which
means periodic misincorporations are not repaired.
After a misincorporation has taken place, the enzyme
will either continue to incorporate nucleotides, causing
a processive mistake, or a terminal event will occur
and elongation will be arrested. Long and Accurate
(LA) PCR combines a highly processive thermostable
polymerase with a second thermostable polymerase
that exhibits a 3’→5’ exonucleolytic activity. This blend
increases the length of amplification products by using
the proofreading polymerase to repair terminal
misincorporations. This repair allows the polymerase
to resume elongating the growing DNA strand.
AccuTaq LA Polymerase Mix combines Sigma’s high
quality Taq DNA polymerase with a small amount of a
thermostable proofreading enzyme. The result is an
enzyme mix that amplifies genomic targets in excess
of 20 kb. Using a less complex template, such as
bacterial genomic and viral DNA, amplifications of up
to 20 kb and 40 kb, respectively, have been achieved.
The fidelity of AccuTaq LA is 6.5 times greater than
Taq DNA polymerase alone.
KlenTaq LA Polymerase Mix is a blend of KlenTaq-1
DNA polymerase (a 5’-exo-minus, N-terminal deletion
of Taq DNA polymerase) and a small amount of a
proof-reading DNA polymerase. This blending of
KlenTaq-1 and a proof-reading polymerase increases
the fidelity, yield and the length of the amplified
product. The usefulness of this blended enzyme is not
limited to long PCR. It works extremely well for DNA
amplifications 0.5-5 kb in length. KlenTaq-1 also has a
broad magnesium optimum, so it is typically
+2
unnecessary to optimize the Mg concentration in the
reaction mixtures. It has a fidelity 4X greater than that
seen in standard Taq DNA polymerase. KlenTaq LA is
suitable for amplifications up to 10 kb in length. It is an
excellent enzyme for amplifying cDNA templates.
Sigma’s Long PCR Core Kit includes all the reagents
necessary to perform long PCR on genomic DNA,
bacterial genomic or viral DNA or cloned DNA
templates with either Sigma’s AccuTaq LA Polymerase
Mix or KlenTaq LA Polymerase Mix.
Reagents
•
10X Buffer for AccuTaq LA, Product
3 x 0.5 ml
No. B0174
500 mM Tris-HCl, 150 mM ammonium sulfate
(pH 9.3, adjusted with KOH), 25 mM MgCl2,
1% Tween 20
•
10X Buffer for AccuTaq LA minus
3 x 0.5 ml
magnesium chloride, Product No. B9923
500 mM Tris-HCl, 150 mM ammonium sulfate
(pH 9.3, adjusted with KOH), 1% Tween 20
C
10X Buffer for KlenTaq LA, Product
1.5 ml
No. B 7673
400 mM Tricine-KOH (pH 9.2 at 25°C), 150 mM
KOAc, 35 mM Mg(OAc)2, 750 :g/ml bovine serum
albumin, acetylated
•
Magnesium Chloride, Product No. M8787
25 mM MgCl2
•
Deoxynucleotide Mix, Product No. D7295 0.25 ml
10 mM dATP, 10 mM dCTP,
10 mM dGTP, 10 mM TTP
•
DMSO, Product No. D8418
•
PCR Water, Product No. W1754
2 x 1.5 ml
•
Control Template, Product No. D8920
2.5 :g/ml lambda DNA
1.5 ml
1 ml
25 :l
•
Control Primer A, Product No. P1343
25 :l
10 pmol/ml Sequence:
5’-ATC ATT ATT TGA TTT CAA TTT TGT CCC
ACT CCC-3'
•
Control Primer B, Product No. P1218
25 :l
10 pmol/ml Sequence:
5'-AGG TCG CCG CCC CGT AAC CTG TCG GAT
CAC CGG AAA-3'
Equipment and Reagents Required But Not Provided
(Sigma Product Numbers have been given where
appropriate)
•
•
•
•
•
•
•
•
•
AccuTaq LA DNA Polymerase Mix, Product No.
D8045
or
KlenTaq LA Polymerase Mix, Product No. D6290
Primers
Sample DNA to be amplified
Dedicated pipets
PCR pipet tips
0.5 ml PCR microcentrifuge tubes, thin walled
Thermocycler
Mineral Oil, Product No. M 8662 (optional)
Precautions and Disclaimer
Sigma's Long PCR Core Kit is for laboratory use only;
not for drug, household or other uses. Warning
statements are included on the label or in the reagents
section of this bulletin where applicable. In addition,
when working with radioactively labeled nucleic acids,
standard procedures for safely handling radioactive
materials should be followed.
Storage/Stability
Store all components except magnesium chloride
solution, DMSO and water below 0ºC. Store the
magnesium chloride solution at 2-8°C. Store the
DMSO and water at room temperature. Frozen DMSO
may be melted at approx. 30ºC without any loss of
performance.
Note: The 10X reaction buffers for AccuTaq LA
contain ammonium sulfate, which can degrade over
time after repeated freeze thaw. This degradation can
compromise the activity of the AccuTaq LA enzyme. If
a drop in activity is observed, discard the buffer
currently being used and open a new vial.
Procedure
For KlenTaq LA Polymerase Mix, see Sigma Technical
Bulletin No. MB-365.
For AccuTaq LA DNA Polymerase Mix, see Sigma
Technical Bulletin No. MB-410.
Technical bulletins including procedures and
references are furnished with AccuTaq LA DNA
Polymerase Mix and KlenTaq LA Polymerase Mix.
Copies are also available from Sigma Technical
Service upon request.
’The PCR process is covered by patents owned by
Hoffman-LaRoche, Inc.
Sigma brand products are sold through Sigma-Aldrich, Inc.
Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications.
Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply.
Please see reverse side of the invoice or packing slip.