User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/08

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Nuclear Gene Adenita Cultures 454 Amplification

A. ricciae

EX Takara RXN Mixture conc 1X 5
Milli Q water 4.15 20.75
Takara EX Buffer 10X 1 5
dNTP 2.5uM 0.8 4
Primer Mix 20uM 1
EX Taq 5U 0.05 0.25
Template Instagene 3 15
45
9.0ul / RXN add 1.0ul Primer
PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Primer
1 A. Ricciae B3
2 A. Ricciae C3
3 A. Ricciae D3
4 A. Ricciae E3
5 A. Ricciae F3
6 Neg Control B3
7 Neg Control C3
8 Neg Control D3
9 Neg Control E3
10 Neg Control F3
11 Pos Control B3
12 Pos Control C3
13 Pos Control D3
14 Pos Control E3
15 Pos Control F3
*Positive Control = AV 20ng

Loaded 2.0ul 08June10.jpg

HCO/LCO Adenita Culture Cycle Sequencing

For failed sequencing sample 14

CS Reaction mixture conc. 1x ' ' AMP Lane Template ' Sample Number Primer
Big Dye Terminator 0.5 1 Ithaca 1 A3 14 HCO
Primer (FWD or RVS) 5uM 0.4 2 Ithaca 1 A3 14 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
60C 4 min
x60