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Method for improving polymerase chain reaction amplification effects by using L-isoleucine

A technology of isoleucine and chain reaction, which is applied in the field of isoleucine to improve the amplification effect of polymerase chain reaction, can solve the problems of inconvenient application and large difference in effect, and achieve improved specificity, obvious effect, Easy to use effects

Inactive Publication Date: 2011-09-14
山东大正医疗器械股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these additives have their own limitations, and the effects vary greatly in different situations, which brings inconvenience to the application.

Method used

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  • Method for improving polymerase chain reaction amplification effects by using L-isoleucine
  • Method for improving polymerase chain reaction amplification effects by using L-isoleucine
  • Method for improving polymerase chain reaction amplification effects by using L-isoleucine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Synergistic effect of isoleucine on general PCR (non-high GC);

[0041] 1) Preparation of isoleucine solution: 10mg / ml

[0042] 2) Sterilization of inosine solution: high temperature sterilization (121°C, 30min)

[0043] 3) Configuration of PCR reaction system:

[0044] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0045] 10X PCR Buffer 1.2μL

[0046] dNTPs (25mM) 0.1μL

[0047] Template (100ng / ul) 0.2μL

[0048] Primer 1 (2.0 μM) 0.75 μL

[0049] Primer 2 (2.0μM) 0.75μL

[0050] Mg 2+ (25mM) 1.2μL

[0051] Taq (5U / μL) 0.2μL

[0052] Isoleucine (10mg / ml) or H 2 O 7.6 μL

[0053]

Primer 1

Primer 2

Amplified length (nt)

1

AGCCAGCTGTTAACCTTGTTCAG

GCCTGATTAAAACCACAGTCACC

963

2

AACCAGCCCAGCACTAATGTTTA

ACACAAATTTAGCCGGACACAGT

941

3

CATAGGAGGGAATCTCCTGGTCT

AGGAAAATGTCTTCAGGGTCTCC

996

[0054] 4

AGGTAAAGGGGACTTTGTCTTGC

CATT...

Embodiment 2

[0065] The synergistic effect of isoleucine on high GC sequence PCR;

[0066] 1) Preparation of isoleucine solution: 10mg / ml

[0067] 2) Sterilization of isoleucine solution: high temperature sterilization (121°C, 30min)

[0068] 3) Configuration of PCR reaction system:

[0069] Configure the system in a thin-walled PCR tube in the following order and dosage.

[0070] 10X PCR Buffer 1.2μL

[0071] dNTPs (25mM) 0.1μL

[0072] Template (100ng / ul) 0.2μL

[0073] Primer 1 (2.0μM) 0.8μL

[0074] Primer 2 (2.0μM) 0.8μL

[0075] Mg 2+ (25mM) 1.2μL

[0076] Taq (5U / μL) 0.2μL

[0077] Isoleucine (10mg / ml) or H 2 O 7.5 μL

[0078]

[0079] Among them, the template is human Genome DNA, which is extracted by our laboratory. Taq enzyme and PCR buffer were purchased from Beijing Sanbo Yuanzhi Bioengineering Company. A total of 12 tubes of PCR system were configured, and 6 pairs of primers were used, numbered 1, 2, 3, 4, 5, and 6; the numbers for adding isoleucine were 1', 2'...

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Abstract

The invention discloses a method for improving polymerase chain reaction (PCR) amplification effects by using L-isoleucine, which relates to the technical field of biology. In the method, the PCR amplification effects are improved by adding a certain amount of the L-isoleucine into a system on the basis of the original PCR amplification method. The method has an obvious optimization amplification effect, is easy and convenient to operate and is easy to master.

Description

technical field [0001] The invention relates to a polymerase chain reaction (PCR) amplification method in the field of biotechnology, in particular to a method for improving the polymerase chain reaction (PCR) amplification effect by isoleucine (L-isoleucine). Background technique [0002] Polymerase chain reaction (PCR), also known as in vitro enzymatic gene amplification, is a gene amplification technology that simulates DNA replication in vitro. This technology was invented by K.Mullis in 1985. Under the action of enzymes and the guidance of specific primers, a large number of gene duplications that only occur when organisms cell division and proliferation can be realized in a test tube in a very short period of time. Generally speaking, within a few hours. Amplify the gene of interest to a million-fold. [0003] Over the past 20 years, PCR technology has gradually matured through continuous progress and development, forming a complete set of technical systems. Conventi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张治洲赵鹤翔张会敏吕志伟王有志
Owner 山东大正医疗器械股份有限公司