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

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Alien Gene LongPCR Sequencing Confirmation

EX Takara RXN Mixture conc 1X 14
Milli Q water 7.15 100.1
Takara EX Buffer 10X 1 14
dNTP 2.5uM 0.8 11.2
Primer Mix 5uM 1 14
EX Taq 5U 0.05 0.7
Template touch colony 0
PCR 1:5 140
PCR cycle
98C 10 sec
60C 1 min
x15

Loaded 2.0ul of sample

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 Terminator 0.5 7.5
Primer (FWD or RVS) 5uM 0.4 6
DMSO 0.1 1.5
5X Seq Buffer 5X 0.4 6
Water 5U/ul 3.6 54
Template * ? 1
75 5.0ul / RXN
* Ethanol Cleaned PCR product
Cycle Seq Program
96C 10 sec
50C 5 sec
60C 4 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 water 14.3 57.2
Takara EX Buffer 10X 2 8
dNTP 2.5uM 1.6 6.4
Primer Mix 5uM 2 8
EX Taq 5U 0.1 0.4
Template touch colony 0
PCR 1:5 80
20.0ul per RXN
PCR cycle
95C 3 min
95C 15 sec
50C 20 sec
72C 1.5 min
x19

Load 2.0ul sample

21July2010B.jpg