User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/13

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LCO/HCO A.vaga amp

EX Takara RXN Mixture conc 1X 1 ' ' AMP Lane Template
Milli Q water 10.3 10.3 1 A. vaga
Takara EX Buffer 10X 2 2
dNTP 2.5uM 1.6 1.6
Primer Mix 20uM 2 2
EX Taq 5U 0.1 0.1
Template 2ng/ul 4
16
16.0ul / RXN add 4.0ul Template
PCR cycle
94C 3 min
94C 10 sec
45-68C 3 min
RAMP 0.5C/sec
x35
68C 5 min

Loaded 2.0ul on Gel

HCO LCO Cycle Sequencing Purification with QIAquick

For A.vaga

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

    • see page 19.

HCO/LCO Adenita Culture Cycle Sequencing

For A.vaga

CS Reaction mixture conc. 1x ' ' AMP Lane Template ' Sample Number Primer
Big Dye Terminator 0.5 1 A.vaga HCO
Primer (FWD or RVS) 5uM 0.4 2 A.vaga LCO
DMSO 0.1
5X Seq Buffer 5X 0.4
Water 0.6
Template * ~5ng/ul 4
2.0ul / RXN
*QIAquick Cleaned PCR product
Cycle Seq Program
96C 10 sec
50C 5 sec

Yeast Culture Prep for Long Term Freezer Storage

From Revived Yeast Culture

Genus species NCYC #
Saccharomyces pastorianus 204
Saccharomyces bayanus 415
Schizosaccharomyces octosporus 427
Saccharomyces kluyveri 543
Saccharomyces paradoxus 700
Saccharomyces dairenensis 777
Saccharomyces exiguus 814
Saccharomyces unisporus 971
Saccharomyces cerevisiae 1022
Saccharomyces servazzii 2307
Schizosaccharomyces japonicus 2355
Saccharomyces rosinii 2827
Saccharomyces barnettii 2878
Saccharomyces mikatae 2888
Saccharomyces kudriavzevii 2889
Saccharomyces cariocanus 2890
Saccharomyces spencerorum 2991
Saccharomyces humaticus 3083
Saccharomyces yakushimaensis 3084

1) Overnight culture on agar plate (or grow until sufficient lawn)

Streaked out 50.0ul of growing culture onto a glucose+yeast extract plate

S. pombe - failed to grow - redo S. cerevisiae - good lawn

2) Prepare freezing media = 20% gycerol in sterile YM media. Aliquot into small freezer tube (1000ul).

3) Collect lawn with sterile toothpick and swirl in freezing media

4) Slow freeze in -30C

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