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

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Contents

Alien Gene 454 Adapter Extension

EX Takara RXN Mixture conc 1X 24 ' ' ' ' ' ' '
Milli Q water6.15147.6
Takara EX Buffer10X124
dNTP2.5uM0.819.2
Primer Mix20uM1
EX Taq5U0.051.2
TemplateOleg PCR 1:101
PCR 1:5192
8.0ul / RXN add 1.0ul Primer and 1.0ul PCR template
PCR cycle
98C10 sec
60C1 min
x15
PrimerColonies ----->283251545564656770
BHist F1/Hok = 1? = 5ok = 10ok = 13ok = 16ok = 20
CHistone 2ok = 4ok = 8ok = 9ok = 17
DHSP82 F2/R2ok = 2ok = 6ok = 11ok = 14ok = 18ok = 21
EMyosin F2/R2
FRhook = 3? = 7ok = 12? = 15ok = 19ok = 22
Primer Set4836745710

Load 2.0ul on gel

Image:08July2010.jpg

Library preparation by Nextera DNA sample Prep kit

Tagmentation Reaction

Reaction mixture conc. '
AGM15.6 ng/ul1.5
Nextera Reaction Buffer LMW5x 2
Nextera Enzyme mix (Roche Titanium-compatible)??0.5
H2O6
total10
55C 5min
Purified by MinElute kit (qiagen)11 ul of buffer EB

Addition of emPCR-Compatible Sites and Library Enrichment

Reaction mixture conc. '
Recovered DNA fragment library??2.5
Nextera PCR buffer2x12.5
Nextera Primer Cocktail50x0.5
Nextera Adaptor150x0.5
Nextera PCR enzyme2.5 U/ul0.5
H2O8.5
total25
72C3 min
95C30 sec
95C10 sec
55C30sec
72C3min
15 cycle

laod 5.0 ul of reaction

Image:0151.jpg

Tagmentation Reaction

Reaction mixture conc. '
AGM15.6 ng/ul0.75
Nextera Reaction Buffer LMW5x 2
Nextera Enzyme mix (Roche Titanium-compatible)??0.5
H2O6.75
total10
55C 5min
Purified by MinElute kit (qiagen)11 ul of buffer EB

Addition of emPCR-Compatible Sites and Library Enrichment

Reaction mixture conc. '
Recovered DNA fragment library??2.5
Nextera PCR buffer2x12.5
Nextera Primer Cocktail50x0.5
Nextera Adaptor150x0.5
Nextera PCR enzyme2.5 U/ul0.5
H2O8.5
total25
72C3 min
95C30 sec
95C10 sec
55C30sec
72C3min
18 cycle

laod 5.0 ul of reaction

Image:0153.jpg

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