User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/21

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Contents

Alien Gene LongPCR Sequencing Confirmation

EX Takara RXN Mixture conc 1X 14
Milli Q water7.15100.1
Takara EX Buffer10X114
dNTP2.5uM0.811.2
Primer Mix5uM114
EX Taq5U0.050.7
Templatetouch colony0
PCR 1:5140
PCR cycle
98C10 sec
60C1 min
x15

Loaded 2.0ul of sample

Image:21July2010A.jpg

Alien Gene LongPCR Purification with QIAquick

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

    • see page 19.


Alien Gene LongPCR Cycle Sequencing

CS Reaction mixture conc. 1x 15 '
Big Dye Terminator0.57.5
Primer (FWD or RVS)5uM0.46
DMSO0.11.5
5X Seq Buffer5X0.46
Water5U/ul3.654
Template *?1
755.0ul / RXN
* Ethanol Cleaned PCR product
Cycle Seq Program
96C10 sec
50C5 sec
60C4 min
x60

Long Term Storage Yeast Cultures - Revived from Freezer

Cultures 1001 and 2722

1) Thaw out one tube of culture in glycerol and YM media 2) 50ul streaked on agar (YE + Glucose) plate 3) Incubate at 33C overnight


Results = Good lawns from overnight culture

--> continue preparing long term storage with remaining yeast cultures

Virus 20S

Amplify Colonies 1, 2 and 4

EX Takara RXN Mixture conc 1X 4
Milli Q water14.357.2
Takara EX Buffer10X28
dNTP2.5uM1.66.4
Primer Mix5uM28
EX Taq5U0.10.4
Templatetouch colony0
PCR 1:580
20.0ul per RXN
PCR cycle
95C3 min
95C15 sec
50C20 sec
72C1.5 min
x19

Load 2.0ul sample

Image:21July2010B.jpg

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