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Taq DNA Polymerases (Native, Recombinant & PlatinumĀ®)
And PlatinumĀ® Taq Antibody
TABLE OF CONTENTS
PRODUCT DESCRIPTION
Native and Recombinant Taq DNA Polymerase
Features and Benefits
Applications
PlatinumĀ® Taq DNA Polymerase & PlatinumĀ® Taq antibody
Features and Benefits
Unit Definitions
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Native/Recombinant Taq DNA Polymerase
Hot Start Protocol
Protocol for making strand specific probes by linear amplification labeling
PlatinumĀ® Taq DNA Polymerase & PlatinumĀ® Taq Antibody
Protocol
Applications notes/Miscellaneous notes
Reaction Parameters and Enzyme Details
Effect of glycogen
Notes on Fidelity
Template sizes tested
Using Taq as an RT enzyme
Head to head with and without antibody
Plat Taq Vs Elongase
Betaine/DMSO
In Situ PCR
Incorporation of modified Oligos into DNA by Taq
Troubleshooting
Nonspecific Bands After PCR (smearing)
Low Yield of Desired Fragment
No Bands
High Rate of Misincorporation
High Molecular Weight, EtBr Stainable Material Left in Wells
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
ASSOCIATED PRODUCTS
RELATED TECHNICAL SUPPORT NOTES
PRODUCT DESCRIPTION
Native and Recombinant Taq DNA Polymerase
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Taq DNA Polymerase (EC 2.7.7.7) is a 832 amino acid single subunit enzyme with a MW of 94,000 [Lawyer (1989)
JBC 264, 6427] and a specific activity of 100,000 U/mg of pure protein with one unit corresponding to 50 fmol of
enzyme. It probably does not contain disulfide bridges since the unreduced enzyme has the same mobility in SDS gels
as the reduced enzyme.
Native Taq Polymerase is purified from the Thermophile, Thermus aquaticus YT1.
Recombinant Taq is produced from the Taq polymerase gene cloned in a pUC-type plasmid conferring ampicillin
resistance and is purified from E. coli. This procedure separates a High Molecular Weight form of the enzyme (94,000)
from a Low Molecular Weight form of the enzyme (87,000); thus Recombinant Taq DNA Polymerase only contains the
94,000 form. This 94,000 form is much more stable and has an associated slight 5' to 3' Exonuclease activity.
The recombinant Taq DNA Polymerase expressed in E. coli showed essentially identical characteristics to native Taq
from Thermus aquaticus with respect to activity, specificity, thermostability and performance in PCR.
The recombinant Taq sequence is identical to the native sequence (this sequence is available from GenBank).
Both will leave a 3' dA overhang on ~30% of the ends of PCR fragments. These enzymes catalyze the incorporation of
dNTPs into DNA using a DNA template, a primer terminus and the divalent cation Mg++. They have a polymerization
dependent 5'-3' exonuclease activity,. The enzymes do not have a significant 3'-5' exonuclease activity and thus no
proof reading function, hence the lower fidelity in comparison to Taq blends and Pfx.
Features and Benefits:
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PCR products up to 6kb.
Choice of Native or Recombinant
Use of for difficult templates
Applications:
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Taq DNA Polymerase is used for:
PCR, 3' RACE, 5' RACETaq is stable up to 95Ā°C; thus it is not necessary to replenish the enzyme in a 35 cycle PCR.
The maximal enzyme activity is between 70-75Ā°C which minimizes secondary structure of the template and results in
high polymerization yield. Annealing temps can be chosen from 30-70Ā°C. Taq can be used to generate PCR products ~6
kb.
Cycle sequencing. It gives more uniform bands than Klenow. The high temperature extension and cycling permits
difficult sequences (hairpins) to be sequenced more readily. It can read through homopolymer sequences (i.e. dG:dC)
better than Klenow. [Murray (1989) NAR 17, 8889]. Taq DNA Polymerase will incorporate boronated-nucleotides into a
PCR product. [Porter (1997) NAR, vol 25, no. 8, pp 1611]
Run Off Synthesis (i.e. Cycle Labeling). Making high specific activity single strand specific probes. [Sturzl (1990) Anal.
Bioch. 185, 164.
PlatinumĀ® Taq DNA Polymerase & PlatinumĀ® Taq antibody
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PlatinumĀ® Taq DNA Polymerase is a 1:1 mixture of Recombinant Taq DNA polymerase: monoclonal antibodies to Taq DNA
polymerase. This results in a DNA polymerase which is activated in a temperature dependent manner (at 94oC) during the
start of PCR. Once dissociated, the Taq DNA polymerase regains its full activity. The Platinum Taq antibody is a mixture of
monoclonal antibodies ready to be complexed.
Because the PlatinumĀ® Taq antibodies bind to Taq and render it inactive until it is denatured at 94oC, its major
application is Hot Start PCR. It is important however to remember that mis-priming occurs at the start of PCR and
because the PlatinumĀ® Taq inactivates the Taq, mispriming will not occur. The denaturation starts at 58 degrees or
higher.
Features and Benefits:
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Broader range of magnesium tolerance.
Less need to optimize the PCR (e.g., [Mg], annealing temperature, primer concentration, etc.).
Increased yield of PCR product.
Allows the assembly of the PCR reactions at room temperature.
Less background, nonspecific PCR products including reduced primer dimer products.
Increased sensitivity in the sense that less target is required.
Unit Definititions:
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One unit of PlatinumĀ® Taq DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in
30 min at 74oC.
1 unit per reaction is sufficient for most reactions/applications, but in some instances it may be necessary to use more
enzyme.
One unit of PlatinumĀ® Taq Antibody is the amount of product required to inhibit one unit of Taq DNA polymerase from
incorporating deoxyribonucleotide into acid-precipitable material.
SHIPPING CONDITIONS
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Primary Secondary
Native Taq DNA Polymerase Dry Ice Wet Ice
Recombinant Taq DNA Polymerase Dry Ice Wet Ice
PlatinumĀ® Taq DNA Polymerase Dry Ice Wet Ice
PlatinumĀ® Taq Antibody Dry Ice Wet Ice
STORAGE CONDITIONS
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Native/Recombinant Taq DNA Polymerase
The recommended storage condition is -20Ā°C. Avoid Room Temperature and repeated freeze/thaws.
Storage buffer:
Taq DNA Polymerase (Native/Recombinant): 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5%
NP40, 0.5% Triton X-100. Supplied at 5U/Āµl.
PlatinumĀ® Taq DNA Polymerase/ PlatinumĀ® Taq Antibody
Store at ā?20C in a non-frost-free freezer.
Storage buffer for PlatinumĀ® Taq as well as PlatinumĀ® Taq antibody is [20 mM TrisCl 8.0, 40 mM NaCl, 2 mM
NaPhosphate, 1 mM DTT, 0.1 mM EDTA, stabilizers, 50% glycerol] plus Stabilizers.
STABILITY
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Native/Recombinant Taq DNA Polymerase
The enzyme is stable 48 months at -20Ā°C and 3 days at 0Ā°C. The enzyme is stable to initial freezing on dry ice. The
enzyme can be diluted in 10 mM Tris (pH 7.6), 0.5 mg/ml BSA, 0.1 mM DTT, 0.1 mM EDTA. Use diluted enzyme the
same day.
Stability during Reaction
Taq Polymerase Half Life Temperature
5 min 97.5Ā°C
40 min 95Ā°C
120 min 92.5Ā°C
Taq Polymerase in a 30 cycle reaction (94Ā°C for 1', 55Ā°C for 1.5 to 2', 72Ā°C for 1.5 to 3') retains 100% activity as defined by
a unit assay and by a second round of PCR with different primers and template with a resultant PCR product after 30
additional cycles. PCR performed in a PE 480.
PlatinumĀ® Taq DNA Polymerase/ PlatinumĀ® Taq Antibody
The polymerase and antibody should be stable a minimum of 28 months if stored properly at -20oC in a non frost-free
freezer.
The antibody can block Taq activity up to 4 days at 37Ā°C (Focus 20.1, 17).
Once dissociated by treatment of the Ab/enzyme mixture at 94Ā°C for 2 min, the Taq DNA polymerase regains its
full activity.
The antibody is inactivated essentially in less than 1 minute at 94Ā°C as judged by the reactivation of Taq DNA
polymerase when PlatinumĀ® Taq was heated for 1 minute (See Figure 2 in Focus 20.1, 18).
We have not tested stability at 4Ā°C. However, we know that the enzyme is stable 28 months at -20Ā°C and 3
days at 0Ā°C.
QC SPECIFICATIONS
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Native/Recombinant Taq DNA Polymerase
Unit range must 4.5-5 U/Ī¼l. (Sold at 5.0 U/Ī¼l)
Single-Stranded Endonuclease: <20% degradation of ĻX174 + strand 60 fold excess of enzyme
Double-Stranded Endonucleases: <40% conversion to form II 60 fold enzyme excess
5' Double-Stranded Exonuclease: <1.0%
3' Double-Stranded Exonuclease: <0.3%
SDS gel electrophoresis: >90% of protein in 94 kDa MW species
PCR Functional Assay. An 800 bp band should be observed from at least 10 ng of human K562 DNA when
using BDNF primers in a 35 cycle PCR reaction. No fragments are seen in the "No DNA" Control Lane.
PlatinumĀ® Taq DNA Polymerase
Platinum Taq DNA Polymerase is evaluated in a DNA polymerization activity assay that measures the percent of Taq
DNA polymerase inhibition versus an uninhibited control. PlatinumĀ® Taq DNA Polymerase is functionally tested for
amplification. The product is tested for the absence of double and single stranded endonuclease activity as well as the
absence of 5'and 3' exonuclease activity
PlatinumĀ® Taq Antibody
PlatinumĀ® Taq Antibody is evaluated in a DNA polymerization activity assay that measures the percent of Taq DNA
polymerase inhibition versus an uninhibited control
PlatinumĀ® Taq Antibody is functionally tested for amplification. The product is tested for the absence of double and
single-stranded endonuclease activity as well as the absence of 5'-and 3'-exonuclease activity.
PROTOCOL AND APPLICATION NOTES
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(back to Protocol and Application Notes)
Native/Recombinant Taq DNA Polymerase
Hot Start Protocol
Protocol for making strand specific probes by linear amplification labeling
PlatinumĀ® Taq DNA Polymerase & PlatinumĀ® Taq Antibody
Protocol
Applications notes/Miscellaneous notes
Reaction Parameters and Enzyme Details
Effect of glycogen
Notes on Fidelity
Template sizes tested
Using Taq as an RT enzyme
Head to head with and without antibody
Plat Taq Vs Elongase
Betaine/DMSO
In Situ PCR
Incorporation of modified Oligos into DNA by Taq
Troubleshooting
Nonspecific Bands After PCR (smearing)
Low Yield of Desired Fragment
No Bands
High Rate of Misincorporation
High Molecular Weight, EtBr Stainable Material Left in Wells
Native/Recombinant Taq DNA Polymerase
Hot Start Protocol:
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The following protocol represents a starting point for any PCR amplification. Optimal incubation times, annealing
temperatures, amount of Taq, amount of primer, MgCl2, and amount of template may need to be varied for the sequence
being amplified.
Add the following to a 0.5 ml Ī¼centrifuge tube on ice.
10X PCR Buffer 10 Āµl
10 mM dNTP Mix 2 Āµl
50 mM MgCl2 3 Āµl
Primers (@ 10 Ī¼M) 5 Āµl each
Template 1-20 Āµl
Water To 100 Āµl
Mix contents of tube and overlay with 50 Ī¼l mineral or silicone oil
Centrifuge briefly to collect contents on bottom.
Incubate tubes in a Thermocycler @ 94Ā°C for 3' for initial template denaturation.
Maintain reaction at 80Ā°C for "Hot Start".
Add 0.2-0.5 Ī¼l (1.0-2.5 U) Taq to each reaction, below the surface of the oil!
Perform 25-35 cycles of PCR as follows:
Denature 94Ā°C for 45s
Anneal 55Ā°C for 30s
Extend 72Ā°C for 90s
Incubate for an additional 10 minutes at 72Ā°C. Soak reaction at 4Ā°C. Store reaction products at -20Ā°C.
PlatinumĀ® Taq DNA Polymerase & PlatinumĀ® Taq Antibody
Protocol:
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PlatinumĀ® Taq DNA Polymerase can be used in the same manner as Taq DNA polymerase! For example, if you have a
PCR protocol that calls for 2.5 units of Taq DNA polymerase, use 2.5 units of PlatinumĀ® Taq DNA Polymerase.
The protocol below can be used at a point of reference with the following change - Ignore steps 1 and 2 since the Taq
and Antibody are pre-complexed. Use only 0.5 Āµl (1.0 unit) of the PlatinumĀ® Taq DNA Polymerase.
The following general procedure is suggested as a guideline and as a starting point when using PlatinumĀ® Taq Antibody
in any PCR amplification. Optimal reaction conditions (incubation times and temperatures, concentration of Taq DNA
polymerase, primers, MgCl2, and template DNA) vary and may need to be optimized. Final reaction volumes described
below are approximately 50 Āµl. Reaction size may be altered to suit user preferences.
1. Measure out the amount of Taq DNA polymerase needed. When using a reaction cocktail, determine the total number
of units in the mix.
2. Add an amount of "equivalent units" of PlatinumĀ® Taq Antibody to an equal amount of Taq DNA polymerase units.
For Taq DNA polymerase at 5 unit per ul, prepare a 1:1 mixture. Mix well using a Vortex Mixer. When complexing the
antibody to the Taq polymerase, no incubation time is needed. The antibody is supplied at such a unit concentration that
the "inactivation of the polymerase" is immediate!
3. Add the following components to a sterile 0.5 ml microcentrifuge tube:
Components Volume Final Concentration
10X PCR Buffer, Minus Mg 5 Āµl 1X
10 mM dNTP mixture 1 Āµl 0.2 mM each
50 mM MgCl2 1.5 Āµl 1.5 mM
Primer mix (10 ĀµM each) 1 Āµl 0.2 ĀµM each
Template DNA >1 Āµl as required
PlatinumĀ® Taq Antibody/ Taq DNA 0.2 Āµl 1.0* units
polymerase
Autoclaved, distilled water to 50 Āµl Not applicable
*1.0 unit is sufficient for amplifying most targets. In some cases, more enzyme may be required (up to 2.5 units).
If desired, a master mix can be prepared for multiple reactions to minimize reagent loss and to enable accurate pipetting.
4. Mix contents of the tubes and overlay with 50 Āµl of mineral or silicone oil, if necessary.
5. Cap the tubes and centrifuge briefly to collect the contents.
6. Incubate tubes in a thermal cycler at 94oC for 30s to 2 min to completely denature the template and activate the enzyme.
7. Perform 25-35 cycles of PCR amplification as follows:
Denature 94oC for 30s
Anneal 55oC for 30s
Extend 72oC for 1 min per kb
8. Maintain the reaction at 4oC after cycling. The samples can be stored at -20oC until use.
Applications notes/Miscellaneous notes:
Reaction Parameters and Enzyme Details:
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OPTIMUM pH Active from pH 7.5-9.5. Unit assay done at 9.3 in TAPs buffer. Sequencing
should be done at 8.3-9.0.
DIVALENT CATION 1-5 mM Mg++, 2 mM Optimum
SALT EFFECTS Stimulated by increasing KCl up to 50 mM. NaCl does not show this effect.
TEMPERATURE Relative rates: 72Ā°C = 100%, R.T. = 0.4%, 37Ā°C = 2.5,
55Ā°C = 40%, 85Ā°C = 40%.
RATE Very processive enzyme. It has an extension rate of 35-100
nt/sec at 75Ā°C [Wittwer (1991) BioTechniques 10(1), 76.
ERROR RATE See Comments Below
INHIBITORS Bromophenol Blue [Wittwer(1991) BioTechniques 10.1, 82]. Urea, DMSO
DMF,formamide and SDS all inhibit Taq to some extent. KCl at
concentrations greater than 75 mM.
COMPATABILITY Xylene Cyanol (0.02%), Tartrazine and Ficoll (2.5%) do not inhibit this
enzyme.
Km for DNA 1.5 nM
Km for dNTPs 15 ĀµM (from NEB Transcript 4.1)
PROCESSIVITY 40 nts per polymerase binding event. (From NEB Transcript 4.1)
EFFECT OF DMSO, Detergents Traditionally DMSO was used for PCR specificity or yield enhancement for
sometargets. DMSO helps to loosen nucleic acid paring so quite often it was
used forsouthern blot hybridization. There are also other types of co-solvents
available such as detergents, Betaine, Betaine+DMSO and PCRx enhancer.
Invitrogenās PCRx enhancer is a different kind of co-solvent than Betaine or
DMSO. Taq is inhibited 47% at a concentration of 10% DMSO. It can still
be used for PCR & Cycle Sequencing of GC rich DNA in the presence of
10% DMSO if the amount of Taq is doubled. Sun(1993) BioTechniques 15.3,
372.
Reaction Rate ~35-150 nucleotides/second.
MOLECULAR WEIGHT: (from Enzymes of Molecular Biology)
Theoretical mass 93,900 Da
Number of amino acids 832
SDS apparent MW 97,300
DOMAINS: #1-410
5'-3' exo #410-832
Polymerase
Effect of glycogen:
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Glycogen was found to have no significant effect on genomic DNA amplification with Taq. Glycogen amounts ranged from
20 ng - 0.01 Āµg per amplification reaction. Genomic DNA levels were at either 5, 10 or 50 ng, typical for a genomic
amplification reaction
Notes on Fidelity:
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(back to Protocol and Application Notes)
See also http://www.nwfsc.noaa.gov/protocols/taq-errors.html.
The error rate reported for Taq is very much dependent on the assay used. Different error rates reported in the literature (and
the corresponding reference citation are listed below):
A 1.1 x 10-4 base substitutions/bp Tindall, et. al. (1988) Biochemistry 27, 6008.
Assay = Reversion of Opal Suppression in LacZ.
B 2.4 x 10-5 frameshift Tindall, et. al. (1988) Biochemistry 27, 6008.
mutations/bp
C 2.1 x 10-4 errors/bp Keohavong, et. al. (1989) PNAS 86, 9253.
Assay = Denaturing Gradient Gel Electrophoresis.
D 7.2 x 10-5 errors/bp Ling, et. al. (1991) PCR Methods Appl. 1 (1), 63.
E 8.9 x 10-5 errors/bp Cariello, et. al. (1991) Nucleic Acids Research 19 (15), 4193.
Assay = DGGE
F 2.0 x 10^-5 errors/bp Lundberg, et. al. (1991) Gene 108, 1.
Assay = Loss of LacI function.
G 1.1 x 10-4 errors/bp Barnes, et. al. (1992) Gene 112, 29.
Assay = Loss of LacZ function.
"Errors/bp" indicates total errors (base substitutions, frameshifts, etc).
The presence of manganese (Mn++) will significantly decrease the fidelity of Taq DNA Polymerase and should be
avoided Imbalances in dNTPs result in decreased fidelity of polymerization. See these references:
PCR Methods and Applications (1992) V2 pp 28-33
Technique (1989) V1 pp 11-15
PCR Methods and Applications (1991) V1 pp 17-24 Eckert & Kunkel
Protein Engineering (1996) V9 pp 77-83
Nature Biotechnology (1996) V14 pp 458-467
PNAS (1993) V90 p5618 K. Chen & F.H. Arnold
Template sizes tested:
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With human genomic DNA templates and different B globin primer sets, 194 bp up to 4.1 kb have been amplified with
better specificity and higher yields.
Using Taq as an RT enzyme:
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(back to Protocol and Application Notes)
Taq has RT activity but its activity is extremely low and can't be detected easily under normal PCR reaction conditions. If
you use Mn instead of Mg, Taqās RT activity can be increased somewhat. However, its RT activity is 100-1000 fold lower
compared to other thermostable DNA polymerases such as Tth DNA polymerase. The Tth DNA polymerase can be used
for cDNA synthesis instead of RT enzymes in the presence of Mn but its cDNA synthesis capacity is limited to relatively
small mRNA targets.
Head to head with and without antibody:
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With human genomic DNA and a primer set that yields a 4.1 kb band for B globin DNA, background bands started
occurring at 1.4 mM MgCl2 when the PlatinumĀ® Taq Antibody was NOT present. When it was present, the non specific
bands did not start occurring until 2.2 mM MgCl2!!
Betaine/DMSO:
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We have tried using betaine for amplifying high G-C rich templates with PlatinumĀ® Taq: betaine up to 2 M has no effect on
the antibody (however, this concentration of betaine will inhibit the Taq DNA polymerase). The recommended betaine
concentration is 0.5-1 M. We recommend using twice as much enzyme when using betaine in the reaction.
Use of 10% DMSO or 20% Glycerol will not affect the antibody. Therefore these additives are fine with PlatinumĀ® Taq.
In Situ PCR:
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Use of PlatinumĀ® Taq in in situ PCR has not been tested. The antibody will contribute significantly to the size of the Taq,
and it is not known whether this will interfere with the ability of the Taq to get into the cells.
Incorporation of modified Oligos into DNA by Taq:
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Taq can incorporate Biotin, Oregon Green 9 and Alexa 488 modified dNTPs.
See: Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA
Taurai Tasara, Bernhard Angerer, Martine Damond, Holger Winter, Sabine, Ulrich Hubscher and Mario Amacker* Nucleic
Acids Research, 2003, Vol. 31, No. 10 2636 ā? 2646.
Troubleshooting:
1. Nonspecific Bands After PCR (smearing)
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The [enzyme], [primer], [Mg2+] and/or [dNTP] was too high.
The annealing temp. was too low for the primers being used.
Too many cycles were used.
The annealing and extension times were too long.
Bad or old primers.
Too much template was used initially. Try to start with 104-106 molecules.
Is the customer trying to amplify directly from a lambda plaque? If so, the plaque may have been "resuspended" in
SM buffer which contains 8 mM MgCl2. Depending on the volume that the "SM resuspended phage" represented in
the final PCR volume, the final [Mg] in the reaction could be higher than what the customer thought.
Consider using additives or e.g. PCR optimizer kit
2. Low Yield of Desired Fragment
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Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that
this technique can inhibit PCR. [Lee (1995) BioTechniques 18, 225].
Not enough enzyme was used.
Denaturation/extension temp. was too high and enzyme died prematurely.
Too much DMSO (>10%).
Incorrect annealing temperature.
Too few cycles.
Insufficient or too much Mg++.
Poorly designed primers.
Carry over inhibitors (esp. blood, serum)
Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.
Not a long enough extension time was used depending on the size of product being amplified.
Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product
[Dohner (1995) Biotechniques 18.6, 964].
Template had long runs of G/C's. [Woodford, et al. in Nucleic Acids Research (1995) 23.3, 539 show that by
eliminating all potassium from the amplification reactions, GC rich regions in templates are sufficiently destabilized
to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6-8% DMSO or 5% DMSO with 1.2-1.8 M
betaine can be used to amplify GC rich templates [See Baskaran (1996) Genome Research 6, 633].
Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin). Add BSA to the PCR
(~160-600 Āµg/ml), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor.
[The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, Fatty
Acid Free, Alcohol precipitated BSA, or Fraction V BSA all should be effective].
3. No Bands
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NaCl at 50 mM will inhibit the enzyme.
Too much KCl in the reaction. Do not exceed 50 mM.
Incorrect annealing temp. was used.
Incomplete denaturation [Time and temp must be long and high enough].
Template had long runs of G/C's. [Woodford, et al in Nucleic Acids Research (1995)23.3, 539 show that by
eliminating all potassium from the amplification reactions, GC rich regions in templates are sufficiently destabilized
to allow PCR].
10% DMSO partially inhibits Taq.
Hemin (in blood samples) inhibits Taq.
Use of super-irradiated (treated with >2500mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product
[Dohner (1995) Biotechniques 18.6, 964]
Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that
this technique can inhibit PCR. [Lee (1995) BioTechniques 18, 225].
Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the
amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor.
4. High Rate of Misincorporation
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[dNTP's] < 1 ĀµM or > 200 ĀµM and were not present at the same conc. Relative to one another.
Imbalanced dNTPs result in decreased fidelity of polymerization. See these references:
PCR Methods and Applications (1992) V2 pp 28-33
Technique (1989) V1 pp 11-15
PCR Methods and Applications (1991) V1 pp 17-24 Eckert & Kunkel
Protein Engineering (1996) V9 pp 77-83
Nature Biotechnology (1996) V14 pp 458-467
PNAS (1993) V90 p5618 K. Chen & F.H. Arnold.
5. High Molecular Weight, EtBr Stainable Material Left in Wells
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(back to Protocol and Application Notes)
This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction.
Try heating to 65Ā°C and putting sample on ice before loading.
ALTERNATE PRODUCTS AND COMPATIBILITY
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Taq DNA Polymerase, Recombinant 10342053, 10342020, 10342129,1034012
Taq DNA Polymerase, Native 18038018, 18038042, 18038067
TaqPCRx Recombinant DNA Polymerase 11508017
Elongase Enzyme Mix 10480010, 10480028
PlatinumĀ® Taq DNA Polymerase 10966018, 10966026, 10966034
PlatinumĀ® Taq DNA Polymerase, High Fidelity 11304011, 11304029
PlatinumĀ® Pfx DNA Polymerase 11708013, 11708021, 11708039
PlatinumĀ® Taq Antibody 10965010, 10965028
PCRx Enhancer System 11495017
PCR SuperMix 10572014
PlatinumĀ® PCR SuperMix 11306016
PCR SuperMix High Fidelity 10790020
PlatinumĀ® Quantitative PCR SuperMix UDG 11730017
PlatinumĀ® TaqPCRx DNA Amplification System 11509015
PCR Reagent System 10198018
Elongase Amplification System 10481018
PRODUCT DOCUMENTATION
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Brochures Citations Cell lines
COA FAQ Licensing
Manuals MSDS Newsletters
Tools & Tips Vector Data
REFERENCES
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Native/Recombinant Taq Polymerase
Chen, et. al. "DNA Polymerase from Thermus aquaticus" (1976) J. Bacteriology 127: 1550.
Innis, et al. "DNA Sequencing with Taq Polymerase" (1988) PNAS 85:9436
Landgraf, et al. "Taq Polymerase; With A Particular Emphasis on Its Use in PCR Protocols". Methods in
Molecular Biology (1993) Humana Press. Chapter 4, 31.
PlatinumĀ® Taq DNA Polymerase/ PlatinumĀ® Taq Antibody
Product profiles for the Platinum Taq DNA Polymerase and the Platinum Taq antibody.
-Focus 19.3, 46."Improved PCR Specificity and Yield with Platinum Taq DNA Polymerase".
-Focus 20.1, 17."Biochemical Characterization of Platinum Taq DNA Polymerase".
PRODUCT NAME AND CATALOG NUMBERS
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Name Size Catalog Number
Native Taq 100 units 18038-018
DNA Polymerase 500 units 18038-042
3 x 500 units 18038-067
5,000 units 18038-240
Recombinant Taq 100 units 10342-053
DNA Polymerase 500 units 10342-020
3 x 500 units 10342-046
5,000 units 10342-178
PlatinumĀ® Taq 100 Reaction 10966-018
250 Reactions 10966-026
DNA Polymerase
500 Reactions 10966-034
5,000 Reactions 10966-083
PlatinumĀ® Taq Antibody 100 Reactions 10965-010
250 Reactions 10965-028
500 Reactions 10965-044
COMPONENTS
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Name Size 50mM MgCl2 10X PCR Buffer Catalog Number
Native Taq 100 units 1 ml 1.25 ml 18038-018
DNA Polymerase 500 units 1 ml 2 x 1.25 ml 18038-042
3 x 500 units 3 x 1 ml 6 x 1.25 ml 18038-067
5,000 units 1 x 10 ml 1 x 50 ml 18038-240
Recombinant 100 units 1 ml 1.25 ml 10342-053
Taq DNA Polymerase 500 units 1 ml 2 x 1.25 ml 10342-020
3 x 500 units 3 x 1ml 6 x 1.25 ml 10342-046
5,000 units 1 x 10 ml 1 x 50 ml 10342-178
PlatinumĀ® Taq 100 Reaction 1 ml 1.25 ml 10966-018
250 Reactions 1 ml 1.25 ml 10966-026
DNA Polymerase
500 Reactions 1 ml 2 x 1.25 ml 10966-034
5,000 Reactions 1 x 10 ml 1 x 50 ml 10966-083
PlatinumĀ® Taq Antibody 100 Reactions 10965-010
250 Reactions 10965-028
500 Reactions 10965-044
10X PCR Buffers: 18067-017
3x1 ml - two vials of 10x PCR buffer (1ml) and one vial of 50mM MgCl2 (1ml)
PlatinumĀ® Taq DNA Polymerase:
Part # 51430. All are in single tube even the 5000 U size is in single tube
[20 mM TrisCl 8.0, 40 mM NaCl, 2 mM NaPhosphate, 1 mM DTT, 0.1 mM EDTA, stabilizers, 50% glycerol]
a. Taq DNA Polymerase/Antibody Mixture. It is supplied at 5 U/Āµl
100 U size of the product is supplied 20 Āµl;
500 U size is 100 Āµl;
5000U is 1ml in a 1.5 ml tube.
b. One vial of 10X PCR Buffer. Part # Y02028. Its composition is 200 mM Tris pH 8.4, 500 mM KCl. 1 ml
supplied. (2 vials of 1 ml are supplied with the 500 U size and 50 ml of 10X PCR Buffer in a 60 ml bottle with the
5000 U size).
c. One vial of 50 mM MgCl2. Part # Y0216. 1 ml supplied. For 5000 U size, 25 ml of MgCl2 in a 30 ml bottle.
PlatinumĀ® Taq Antibody:
This is a single component product. It comes in a 100 unit size at 5 U/Āµl. Therefore 20 Āµl is supplied in [20 mM Tris pH
8.0, 40 mM NaCl, 2 mM NaPhosphate, 1 mM DTT, 0.1 mM EDTA, stabilizers, 50% glycerol] plus Stabilizers
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Property table
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TITLE
KEYWORD
SURVEY
GCMS ID
STELLENT ID
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