Application of Ectoine used as synergist in PCR

A technology of tetrahydropyrimidine and a synergist is applied in the field of tetrahydropyrimidine enhancing PCR amplification efficiency, which can solve problems such as increasing non-specific amplification, and achieve the effects of improving operation, improving PCR specificity and low cost

Inactive Publication Date: 2015-04-29
ZHENJIANG BIO INNOVA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, each of these additives has its limitations. For example, DMSO can inhibit the formation of secondary structures. Therefore, when the content of guanine G and cytosine C is high and the content of adenine A and thymine T is low, adding an appropriate amount of DMSO can improve PCR amplification. Efficiency, when the content of G and C is low, it will inhibit PCR amplification; Tween-20 can make the polymerase more stable, but it also increases non-specific amplification

Method used

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  • Application of Ectoine used as synergist in PCR
  • Application of Ectoine used as synergist in PCR
  • Application of Ectoine used as synergist in PCR

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Experimental program
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Effect test

preparation example Construction

[0013] 1. Preparation of ectoine additive: Weigh 15 mg of commercially available ectoine, place it in a sterilized 1.5ml centrifuge tube, add sterilized water to dissolve it, and finally set the volume to 1ml. 20min sterilization.

[0014] 2. The configuration of the PCR reaction system: the DNA fragments to be amplified are different, and the dNTP, Mg 2+ 1. The amount of template added is different; an appropriate amount of ectoine solution is added to the above reaction system, and the amount of double distilled water added should be deducted accordingly according to the addition of ectoine solution.

[0015] 3. PCR reaction operation: pre-denaturation, denaturation, annealing temperature and time should be selected according to the template, and the extension time should be selected according to the length of the target fragment to be amplified.

[0016] 4. Detection of PCR amplification products: detection by agarose gel electrophoresis.

Embodiment 1

[0017] Example 1: Optimal amplification of ectoine to a 23kb DNA fragment

[0018] 1. Prepare 15mg / ml ectoine aqueous solution.

[0019] 2. Configuration of PCR reaction system:

[0020] 10×PCR Buffer 2.5ul

[0021] dNTP (2.5mM) 5ul

[0022] Primer 1 (2.0uM) 1ul

[0023] Primer 2 (2.0uM) 1ul

[0024] Mg 2+ (25mM) 1.5ul

[0025] After configuring the above reaction system, add an appropriate amount of ectoine additive. 1 in the attached figure means adding 10ul ectoine solution, and add sterilized double distilled water to make up the 25ul system. 2 in the figure means adding 14ul ectoine solution. , the final concentrations of the ectoine solution in the PCR reaction system were 6 mg / ml and 8.4 mg / ml respectively. In the control group, the ectoine solution was replaced with 14ul sterilized double distilled water.

[0026] 3. PCR reaction run:

[0027]

[0028]

[0029] 4. After PCR reaction, use agarose gel electrophoresis to detect: the results are shown in...

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Abstract

The invention aims to provide a PCR synergistic additive Ectoine. The Ectoine can be used to enhance PCR amplification effect and especially has a remarkable effect of amplifying super-long DNA fragments.

Description

technical field [0001] The invention relates to a PCR synergist, in particular to a method for ectoine to enhance PCR amplification efficiency. Background technique [0002] Polymerase chain reaction (referred to as PCR), also known as cell-free molecular cloning, is a molecular biology technique for enzymatically synthesizing DNA in vitro. It consists of several steps of high-temperature denaturation, low-temperature annealing, and moderate extension. , cyclically, the target DNA can be rapidly amplified in a short time. It has the characteristics of strong specificity, high sensitivity, simple operation, and time saving. It can be used not only for basic research such as gene isolation, cloning and nucleic acid sequence analysis, but also for disease diagnosis or wherever there is DNA and RNA. Invented by American scientist Dr.Mullis, Mullis won the 1993 Nobel Prize in Chemistry due to the cross-age significance of PCR technology in theory and application. [0003] In r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 朱道辰陈焱洪好
Owner ZHENJIANG BIO INNOVA BIOTECH
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