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PCR

i-pfu DNA Polymerase

Cat.No Capacity Inquire
25181 250 unit Inquire
PRODUCT INFORMATION
Description

PCR core kit of high-purity pfu DNA polymerase with low error rate and proofreading function that is essential for high-accuracy gene cloning

  • • High accuracy
       - Possible to amplify long DNA template with Proof-reading activity
       - Optimal for experiments requiring accuracy such as gene cloning and expression
  • • Low error rate
       - 3’→5’ Exonuclease is a typical thermostable proof-reading enzyme with an error rate of 1x10-6
  • • Blunt-end Cloning

i-pfu DNA Polymerase is a typical heat-resistant proof-reading enzyme with 3 '→ 5' exonuclease activity. It has high fidelity and guarantees an error rate of 1x10-6 (one error rate per 13,000 base pairs) .The proof-reading activity of pfu DNA Polymerase is that when a DNA polymerase is polymerized, mismatched (non-complementary) nucleotides can be removed and corrected with complementary nucleotides. This activity allows PCR products of more accurate base sequences to be obtained than Taq DNA Polymerase, but allows amplification of longer DNA templates compared to Taq DNA Polymerase. This is because the mismatched nucleotide interferes with the polymerization reaction.

The Pfu DNA Polymerase can remove the DNA polymerase from the template during the DNA synthesis process. It is suitable for long-size PCR (up to ~ 30 kb) which is difficult to amplify. It has high fidelity and proof reading function compared with general Taq DNA Polymerase. As a result, DNA fragments longer than Taq DNA Polymerase (up to 30 kb) can be amplified. Because of these properties, Pfu DNA Polymerase is widely used in experiments where accuracy is required, such as gene cloning and expression, although polymerization efficiency is low and it takes more time to polymerize.Unlike Taq DNA Polymerase, this i-pfu DNA Polymerase produces blunt-end amplification products. Therefore, in order to clone the amplification product, it can be cloned into a blunt-end vector. To clone T vector, a base A must be added.

Applications
  • 01General PCR
  • 02template amplification for DNA sequence analysis
  • 03Blunt Cloning, etc.
Kit Contents
Content 250 Units
i-pfu DNA Polymerase(2.5U/ml) 250 units x 1 tube
10× PCR Buffer(w/15 mM Mg2+) 1 ml x 1 tube
10× Mg2+ free PCR Buffer 1 ml x 1 tube
10 mM dNTPs (2.5 mM/each) 500 μl x 1 tube
25 mM Mg2+ 1 ml x 1 tube
Manual 1 ea

 

 

Technical Data

Sensitivity comparison test

To compare the sensitivity of i-pfu DNA Polymeras and Company A and B using the same function product, 1 Kb and 4.5 Kb fragment primer was diluted by template concentration and amplified. i-pfu DNA Polymeras exhibits excellent sensitivity even at lower template concentrations compared to products with the same function of other companies.

[ Panel A ] 1 Kb amplification(λDNA)
Lane M, 1 Kb DNA Ladder; lane 1, 200 pg λDNA; lane 2, 100 pg λDNA; lane 3, 50 pg λDNA; lane 4, 25 pg λDNA; lane 5, 12.5 pg λDNA; lane 6, 6.25 pg λDNA; lane 7, 3.125 pg λDNA; lane N, negative control

[ Panel B ] 4.5 Kb amplification(TopoXL/5F)
Lane M, 1 Kb DNA Ladder; lane 1, 50 ng DNA; lane 2, 10 ng DNA; lane 3, 2 ng DNA; lane 4, 400 fg DNA; lane 5, 80 fg DNA; lane 6, 16 fg DNA; lane 7, 3.2 fg DNA; lane N, negative control

TroubleShooting Guide
QIn order to perform the experiment, the manual shows only the experimental method when the reaction volume is 50ul. How much should I put taq in trying to change volume to 20ul?
AIf the PCR reaction volume is reduced from 50ul to 20ul, the same amount of Taq should be added. You can experiment with the same amount of template and primer. Do not add 5μl of 10X PCR buffer and reduce to 2μl.
QI am currently performing full genomic sequencing. Do you have a high fidelity product?
AIn case of our i-pfu, the error rate is 1X10-6 and it can be amplified up to 10kb. If you want a product with higher functionality, we recommend the i-StarMAXII™ product, which has an error rate of 1.8X10-8 and can synthesize up to 20kb.
QHIV mutagenesis is under study. point mutation. Is there a suitable product for which kit products are expensive and I want to buy only an enzyme?
Ai-pfu™ DNA polymerase is recommended. i-pfu DNA polymerase has a proof-reading activity that lowers the rate of mutation during the PCR reaction. 
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