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The Construction of Physcomitrella patens C3H Gene Knock Out Vector and Optimum Condition of PCR for C3H1-nptⅡ-C3H2 Target Fragment(PDF)

《北方园艺》[ISSN:1001-0009/CN:23-1247/S]

Issue:
2016年05
Page:
113-118
Research Field:
Publishing date:

Info

Title:
The Construction of Physcomitrella patens C3H Gene Knock Out Vector and Optimum Condition of PCR for C3H1-nptⅡ-C3H2 Target Fragment
Author(s):
CUI Caiyun; YAN Guohong; JIANG Shan
(School of Life Sciences，Guizhou Normal University，Guiyang，Guizhou 550001)
Keywords:
Physcomitrella patens C3H gene; gene knock out; vector construction; annealing temperature
PACS:
-
DOI:
10.11937/bfyy.201605031
Abstract:
Taking Physcomitrella patens as experimental materials,the full gene sequence of C3H that was 1 981 bp in the Physcomitrella patens would be divided into upstream fragment (1 000 bp)，the downstream fragment(981 bp).Primers were designed with upstream and downstream fragment，respectively.The first half was named upstream arm (C3H1，735 bp) and the latter half was called downstream arm (C3H2，700 bp).Using the total DNA of Physcomitrella patens to template and the designed primer of C3H1 and C3H2，amplified C3H1 which was adding restriction site was XhoI and EcoRV and C3H2 which was adding restriction site was NdeI and BamHI by PCR.The C3H1 and C3H2 were inserted into the pTN182 vector and knockout vector(C3H1-pTN182-C3H2 plasmid) was succeed.C3H1-nptII-C3H2 fragment was obtained by PCR，The research of the primer concentration and annealing temperature that had an effect on the concentration and specific of the PCR product was by the single factor method.The results showed that the specificity and concentration of PCR product was higher when primer concentration was 0.20 μmol/L and annealing temperature was 58.7℃.

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Last Update: 2016-04-27