User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/22

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Contents

MATA2 Amplification Yeast Culture

EX Takara RXN Mixture conc 1X 23
Milli Q water2.1549.45
Takara EX Buffer10X123
dNTP2.5uM0.818.4
MATA2 Primer Mix5uM123
EX Taq5U0.051.15
TemplateQiagen Extraction5
115
5.0ul / RXN and 5.0ul Qiagen Extraction
GenusspeciesNCYC #ng/ul
Saccharomycespastorianus2042.65
Saccharomycesbayanus4150.94
Schizosaccharomycesoctosporus4272.27
Saccharomyceskluyveri5431.66
Saccharomycesparadoxus7001.59
Saccharomycesdairenensis7770.94
Saccharomycesexiguus8141.21
Saccharomycesunisporus9711.83
Saccharomycescerevisiae10012.66
Saccharomycescerevisiae10221
Saccharomycesservazzii23071.37
Schizosaccharomycesjaponicus23552.07
Schizosaccharomycespombe27221.95
Saccharomycesrosinii28271.21
Saccharomycesbarnettii28780.63
Saccharomycesmikatae28881.3
Saccharomyceskudriavzevii28893.58
Saccharomycescariocanus28902.01
Saccharomycesspencerorum29912.44
Saccharomyceshumaticus30832.34
Saccharomycesyakushimaensis30841.3
PCR cycle
98C10 sec
60C1 min
x15

Loaded 2.0ul of sample

Image:22July2010.jpg

20S PCR QIAquick PCR clean up and Cycle Seq

http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx#Tabs=t2

Eluted in 30ul of EB buffer

CS Reaction mixture conc. 1x 3
Big Dye Terminator0.51.5
Primer (FWD or RVS)5uM0.41.2
DMSO0.10.3
5X Seq Buffer5X0.41.2
Water0.61.8
Template *~1-3ng/ul4
2.0ul / RXN
*QIAquick Cleaned PCR product
Cycle Seq Program
96C10 sec
50C5 sec
60C4 min
x60
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