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File name:古菌DGGE PCR扩增
Category:Protocols
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Published:2013-12-11 03:40:12
Update:2013-12-11 03:44:21
Summary:

一、试剂

古菌16S rRNA基因V6-V8区DGGE引物

GC-Arch915: CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G AGG AAT

                       TGG CGG GGG AGC AC

UNI-b-rev: GAC GGG CGG TGT GTR CAA

二、实验步骤

1、PCR体系

   10x buffer                           5 μl

   dNTPs (2.5 mM)               4 μl

   GC-Arch915 (20 μM)        1 μl

   UNI-b-rev (20 μM)            1 μl

   rTaq                                0.25 μl

   BSA(1 μg/μl)              33.5 μl

   DNA                            约100 ng

   ddH2O                        补至50 μl

注:BSA可视情况添加

2、PCR程序

      94℃变性3 min;

      94℃变性30 s, 61℃退火30 s,72℃延伸1 min,退火温度在每个循环递减0.5℃,进行 10个循环;

      94℃变性30 s ,56℃退火30 s ,72℃延伸1min ,进行25个循环;

      72℃延伸7 min。

 

参考文献:

1、Yu Z, Morrison M.Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting 

      of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis. Appl Environ Microbiol,

      2004, 8(70):  4800–4806.

2、Yu Z, García-González R, Schanbacher FL, Morrison M. Evaluations of Different Hypervariable Regions of

      Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient

      Gel Electrophoresis. Appl Environ Microbiol, 2008, 3(74): 889–893.

 

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