Name: PCR Kit with Taq
SKU: D7232
Price: 14.00 EUR
Availability: InStock

Description

PCR Kit with Taq provides all reagents required for PCR, except for templates and primers. It is suitable for routine PCR or RT-PCR assays, as well as cloning of DNA fragments that are not too long.

The Taq DNA Polymerase included in this kit is a highly thermostable DNA polymerase derived from Thermus aquaticus with a molecular weight of 94kDa and a half-life greater than 40 minutes when incubated at 95℃. It catalyzes the 5’→3′ polymerization of deoxyribonucleotides in a DNA template-dependent manner. As this product lacks 3’→5′ exonuclease activity and is equivalent to a nucleotide transferase during DNA polymerization, it generates PCR products with 3′ dA overhangs.

Definition of activity: One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 70℃. Enzyme activity is assayed in the following mixture: 67 mM Tris-HCl (pH 8.8 at 25℃), 6.7 mM MgCl₂, 1 mM 2-mercaptoethanol, 50 mM NaCl, 0.1 mg/ml BSA, 0.75 mM activated calf thymus DNA, 0.2 mM of each dNTP, 0.4 MBq/ml [3H]dTTP.

Purity: free of DNA endonuclease and exonuclease, phosphatase, and RNAase.

Enzyme storage buffer: 20 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20 and 50% (v/v) glycerol.

10X PCR Buffer (with Mg²⁺): 100 mM Tris-HCl (pH 8.8 at 25℃), 500 mM KCl, 15 mM MgCl₂, 0.8% (v/v) Nonidet P40.

Inactivation or inhibition: Taq DNA Polymerase can be inactivated by phenol chloroform extraction and inhibited by 0.06% sodium deoxycholate, 0.01% SDS, or 0.02% sarkosyl.

When a PCR reaction volume of 20μl is used, D7232 is sufficient for 400 reactions, respectively.

Packing List：

Item	Component	Quantity
D7232-1	Taq DNA Polymerase (5U/µl)	200U
D7232-2	10X PCR Buffer (with Mg²⁺)	1ml
D7232-3	dNTP (2.5mM each)	0.7ml
D7232-4	6X DNA Loading Buffer	1ml
Manual	–	1 copy

Storage Conditions：

Store at -20℃.

Precautions：

Because PCR reaction is extremely sensitive, contamination must be avoided during preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.

Taq DNA Polymerase in PCR amplification has an error rate of 2.2 x 10-5 errors per nucleotide per cycle. This product is an ideal choice for qualitative or quantitative analysis by regular PCR or qPCR. To clone a DNA fragment longer than 1kb, DNA polymerase with proof-reading activity is recommended, such as Pfu DNA polymerase and BeyoTaq DNA polymerase.

This product is for R&D only. Not for drug, household, or other uses.

For your safety and health, please wear a lab coat and disposable gloves during the operation.

Additional information

Weight	1 kg
Dimensions	30 × 30 × 20 cm

Instruction

Instructions for Use：

1.Prepare PCR reactions:
a. Thaw PCR components at room temperature and mix well prior to use. Keep the Taq DNA Polymerase on ice.
b. Set up the the reaction on ice as follows:.

Component	Final concentration	Volume	Volume
Nuclease-free water	–	(36.75-x)µl	(14.7-y)µl
10X PCR Buffer (with Mg²⁺)	1X	5µl	2µl
dNTP (2.5mM each)	0.2mM each	4µl	1.6µl
Template DNA	10pg-1µg*	xµl	yµl
Primer mixture (10μM each)	0.8µM	4µl	1.6µl
Taq DNA Polymerase (5U/µl)	1.25U/50µl	0.25µl	0.1µl
Total volume	–	50µl	20µl

Note 1: When multiple reactions are required, prepare a master mix including all reagents except template and primer, and then dispense to different nuclease-free PCR tubes. Sometimes, the master mix can also include the primer.
Note 2: The recommended amount of template varies for different types of DNA. In a reaction volume of 50μl, use 0.1-1μg of Mammalian genomic DNA, 10-100 ng of E. coli genomic DNA, or 0.1-10 ng of Plasmid DNA. Too much template DNA can cause non-specific PCR amplifications.
a. Mix the PCR PCR reaction mixture by gentle vortex or pipetting. Centrifuge briefly at low speed to allow liquid to accumulate at the bottom of the PCR tube.
b. Optional) When using a thermocycler without a heated lid, add a drop of mineral oil (ST275) onto the top of PCR reaction mixture.
2.Transfer PCR reactions to a thermocycler and run thermocycling conditions as follows:

Step	Settings	Cycles
Initial denaturation	94℃ 3min	1
Denaturation	94℃ 30sec	30
Annealing	55℃ 30sec
Extension	72℃ 1min
Final extension	72℃ 10min	1
Hold	4℃ forever	–

Note 1: PCR running conditions should be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc.
Note 2: The optimal extension time varies depending on the amplicon length. The recommended extension time is one minute per kb (e.g. 3 minutes for 3 kb amplicons).
Note 3: For initial PCR, the number of cycles can be set to 35 to ensure that the expected PCR product can be amplified. The number of cycles for semi-quantitative or quantitative PCR analysis must be optimized appropriately so that the PCR reaction does not reach a plateau.
FAQ:
1. Few PCR products or no specific bands.
a. It could be due to poor design of primers. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work normally but not your primers, redesign primers.
b. DNA to be amplified may have a high GC content. High GC genes are relatively difficult to be amplified. In such a case, GC-rich buffer suitable for amplifying DNA with high GC content can be used, and PCR reaction parameters should be adjusted accordingly. Direct addition of 1-10% DMSO or 5-20% glycerol is also helpful for amplifying fragments with high GC content.
c. Amplification of long fragments. Although Taq DNA polymerase can amplify DNA fragments up to 8 kb, most of the time it is more suitable for amplification of fragments less than 3 kb.
d. PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.
e. The presence of secondary structure in primers, primer dimers or short primers, may result in poor annealing of primers to the target sequence. In this case, try touch down or other methods for annealing. A gradual cooling from 65℃ to 55℃ or 50℃ is usually used to make annealing more efficiently.
f. The annealing temperature needs to be optimized. If necessary, use a temperature gradient to determine the optimal annealing temperature for each template-primer pair combination.
g. Insufficient extension time. The extension time can be extended 2-5 times from the recommended extension time, and can be set to 5 minutes per 1kb for fragments difficult to amplify.
h. Insufficient denaturation. To amplify long DNA or high GC DNA fragment, the initial denaturation temperature can be adjusted to 95℃ for 1 min or even 95℃ for 2-4 min.
i. Perform PCR reactions on a different thermal cycler to avoid possible problems with the instrument.
j. Insufficient number of PCR cycles. Try more PCR cycles, but do not exceed 40 cycles.
k. Insufficient amount of template. Add more DNA templates or try nested PCR or secondary PCR. Nested PCR is to design another pair of PCR primers inside the original PCR primers, and then conduct PCR amplification again with the diluted first PCR product as template. Instead, secondary PCR uses the same primers for second PCR amplification with the diluted PCR product as template. Nested PCR usually can remove the non-specific DNA amplification, but secondary PCR cannot.
l. DNA sample contains substances that inhibit the PCR reactions. In such a case, template DNA can be purified using appropriate DNA purification methods such as column purification.
m. When non-specific fragments are produced, the annealing temperature can be increased appropriately.
n. Positive and negative controls are always recommended when optimizing PCR reactions.