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PET series carrier forward sequencing primers and sequencing method

A technology of forward sequencing and primer sequence, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc. Experimental cycle, high success rate, and the effect of increased success rate

Inactive Publication Date: 2019-12-27
WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using universal primers for sequencing, the forward sequencing results often have weak or no signal due to low plasmid concentration, resulting in short or unusable sequencing results and a low success rate

Method used

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  • PET series carrier forward sequencing primers and sequencing method
  • PET series carrier forward sequencing primers and sequencing method
  • PET series carrier forward sequencing primers and sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] In this example, the PET series vector containing the target gene was sequenced, wherein the forward sequencing primer used was 5'-GCGAAATTAATACGACTCACTAT-3', the length of the primer was 23bp, and the Tm temperature was 47.4°C.

[0026] The specific sequencing steps are as follows:

[0027] (1) Dilute the primers with ddH2O to a concentration of 5pmole / μl, and configure the following system in a 96-well PCR plate: 1 μl of PET carrier containing the target gene, 1 μl of forward sequencing primer, 2 μl of BigDye mix, ddH 2 O 1 μl;

[0028] (2) Seal the PCR plate with a silica gel pad, put it into a centrifuge and centrifuge it out for a short time, and run it in the PCR instrument according to the following procedures: the PCR program is 96°C for 1min; 96°C for 10s, 50°C for 5s, 60°C for 4min, 25 cycles Cycle; keep warm at 4°C;

[0029] (3) After PCR, take out the 96PCR plate, add 2μl EDTA / NaAC and 20ul cold alcohol to each well, and let stand for 8min;

[0030] (4) S...

Embodiment 2

[0038] In this embodiment, the PET series vector of the target gene is sequenced, wherein the forward sequencing primer used is 5'-RAWWKTAATACGACTCACTAT-3', the length of the primer is 21 bp, and the Tm temperature is 39.5°C.

[0039] The specific sequencing steps are as follows:

[0040] (1) Dilute the primers with ddH2O to a concentration of 3 pmole / μl, and configure the following system in a 96-well PCR plate: 1 μl of PET carrier of the target gene, 1 μl of forward sequencing primer, 2 μl of BigDyemix, ddH 2 O 1 μl;

[0041] (2) Seal the PCR plate with a silica gel pad, put it into a centrifuge and centrifuge it out for a short time, and run it in the PCR instrument according to the following procedures: the PCR program is 96°C for 1min; 96°C for 10s, 50°C for 5s, 60°C for 4min, 25 cycles Cycle; keep warm at 4°C;

[0042] (3) After PCR, take out the 96PCR plate, add 1μl EDTA / NaAC and 12ul cold alcohol to each well, and let stand for 8min;

[0043] (4) Shake gently on a v...

Embodiment 3

[0051] In this example, the PET serial vector of the target gene was sequenced, wherein the forward sequencing primer used was 5'-RAWWKTAATACGACTCACTA-3', the length of the primer was 20 bp, and the Tm temperature was 38.5°C.

[0052] The specific sequencing steps are as follows:

[0053](1) Dilute the primers with ddH2O to a concentration of 7pmole / μl, and configure the following system in a 96-well PCR plate: 1 μl of PET carrier containing the target gene, 1 μl of forward sequencing primer, 2 μl of BigDye mix, ddH 2 O 1 μl;

[0054] (2) Seal the PCR plate with a silica gel pad, put it into a centrifuge and centrifuge it out for a short time, and run it in the PCR instrument according to the following procedures: the PCR program is 96°C for 1min; 96°C for 10s, 50°C for 5s, 60°C for 4min, 25 cycles Cycle; keep warm at 4°C;

[0055] (3) After PCR, take out the 96PCR plate, add 3μl EDTA / NaAC and 24ul cold alcohol to each well, and let stand for 8min;

[0056] (4) Shake gently...

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Abstract

The invention relates to the field of gene sequencing, in particular to PET series carrier forward sequencing primers and a sequencing method. The PET series carrier forward sequencing primers provided by the invention are located on a T7 promoter sequence and have the length of 20-25 bp. The invention further provides the PET series carrier sequencing method which includes the following steps: the forward sequencing primers are respectively adopted to be configured with a sequencing reaction system for PCR, after PCR is completed, EDTA / NaAC and cold alcohol are added into the system for stillstanding, centrifuging and precipitation collecting are conducted, then precipitation is subjected to impurity removing for multiple times through ethyl alcohol, and then after precipitation dissolving, a solution is placed into a sequencing device for sequencing. The PET series carrier forward sequencing primers are high in sequencing success rate, and especially for templates with the low concentration, the success rate can be significantly increased, through the method for sequencing utilizing the forward primers, the success rate is high, the time is saved, and the experimental cycle is shortened.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a PET series carrier forward sequencing primer and a sequencing method. Background technique [0002] In molecular biology research, DNA sequence analysis is the basis for further research and transformation of target genes. Currently, the technologies used for DNA sequencing mainly include the Sanger dideoxy chain termination method (Chain Termination Method) invented by Frederick Sanger, a rapid DNA sequencing method. The emergence of biotechnology has greatly promoted the research and discovery of biology and medicine. The Sanger sequencing method has been widely used in the sequencing technology of different types of samples such as bacteria, plasmids, and PCR products, so as to provide basic data analysis of plasmid extraction, PCR purification, sequencing primer design, and DNA sequence. [0003] The PET system is the most powerful system for cloning and expressing recombina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/113
Inventor 付强华权高李立曾庆耀曹志雄舒芹张雪娇
Owner WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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