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
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 |
|
|
|
|