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

From OpenWetWare

Jump to: navigation, search
Alien genes Main project page
Previous entry      Next entry

Contents

LCO/HCO A.vaga amp

EX Takara RXN Mixture conc 1X 1 ' ' AMP Lane Template
Milli Q water10.310.31A. vaga
Takara EX Buffer10X22
dNTP2.5uM1.61.6
Primer Mix20uM22
EX Taq5U0.10.1
Template2ng/ul4
16
16.0ul / RXN add 4.0ul Template
PCR cycle
94C3 min
94C10 sec
45-68C3 min
RAMP 0.5C/sec
x35
68C5 min

Loaded 2.0ul on Gel

Image:14July2010.jpg

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 Terminator0.51A.vagaHCO
Primer (FWD or RVS)5uM0.42A.vagaLCO
DMSO0.1
5X Seq Buffer5X0.4
Water0.6
Template *~5ng/ul4
2.0ul / RXN
*QIAquick Cleaned PCR product
Cycle Seq Program
96C10 sec
50C5 sec

Yeast Culture Prep for Long Term Freezer Storage

From Revived Yeast Culture

Genus species NCYC #
Saccharomycespastorianus204
Saccharomycesbayanus415
Schizosaccharomycesoctosporus427
Saccharomyceskluyveri543
Saccharomycesparadoxus700
Saccharomycesdairenensis777
Saccharomycesexiguus814
Saccharomycesunisporus971
Saccharomycescerevisiae1022
Saccharomycesservazzii2307
Schizosaccharomycesjaponicus2355
Saccharomycesrosinii2827
Saccharomycesbarnettii2878
Saccharomycesmikatae2888
Saccharomyceskudriavzevii2889
Saccharomycescariocanus2890
Saccharomycesspencerorum2991
Saccharomyceshumaticus3083
Saccharomycesyakushimaensis3084

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

Personal tools