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

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MATA2 Amplification Yeast Culture

EX Takara RXN Mixture conc 1X 23
Milli Q water 2.15 49.45
Takara EX Buffer 10X 1 23
dNTP 2.5uM 0.8 18.4
MATA2 Primer Mix 5uM 1 23
EX Taq 5U 0.05 1.15
Template Qiagen Extraction 5
115
5.0ul / RXN and 5.0ul Qiagen Extraction
Genus species NCYC # ng/ul
Saccharomyces pastorianus 204 2.65
Saccharomyces bayanus 415 0.94
Schizosaccharomyces octosporus 427 2.27
Saccharomyces kluyveri 543 1.66
Saccharomyces paradoxus 700 1.59
Saccharomyces dairenensis 777 0.94
Saccharomyces exiguus 814 1.21
Saccharomyces unisporus 971 1.83
Saccharomyces cerevisiae 1001 2.66
Saccharomyces cerevisiae 1022 1
Saccharomyces servazzii 2307 1.37
Schizosaccharomyces japonicus 2355 2.07
Schizosaccharomyces pombe 2722 1.95
Saccharomyces rosinii 2827 1.21
Saccharomyces barnettii 2878 0.63
Saccharomyces mikatae 2888 1.3
Saccharomyces kudriavzevii 2889 3.58
Saccharomyces cariocanus 2890 2.01
Saccharomyces spencerorum 2991 2.44
Saccharomyces humaticus 3083 2.34
Saccharomyces yakushimaensis 3084 1.3
PCR cycle
98C 10 sec
60C 1 min
x15

Loaded 2.0ul of sample

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 Terminator 0.5 1.5
Primer (FWD or RVS) 5uM 0.4 1.2
DMSO 0.1 0.3
5X Seq Buffer 5X 0.4 1.2
Water 0.6 1.8
Template * ~1-3ng/ul 4
2.0ul / RXN
*QIAquick Cleaned PCR product
Cycle Seq Program
96C 10 sec
50C 5 sec
60C 4 min
x60