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

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Alien Gene 454 Adapter Extension

EX Takara RXN Mixture conc 1X 24 ' ' ' ' ' ' '
Milli Q water 6.15 147.6
Takara EX Buffer 10X 1 24
dNTP 2.5uM 0.8 19.2
Primer Mix 20uM 1
EX Taq 5U 0.05 1.2
Template Oleg PCR 1:10 1
PCR 1:5 192
8.0ul / RXN add 1.0ul Primer and 1.0ul PCR template
PCR cycle
98C 10 sec
60C 1 min
x15
Primer Colonies -----> 28 32 51 54 55 64 65 67 70
B Hist F1/H ok = 1 ? = 5 ok = 10 ok = 13 ok = 16 ok = 20
C Histone 2 ok = 4 ok = 8 ok = 9 ok = 17
D HSP82 F2/R2 ok = 2 ok = 6 ok = 11 ok = 14 ok = 18 ok = 21
E Myosin F2/R2
F Rho ok = 3 ? = 7 ok = 12 ? = 15 ok = 19 ok = 22
Primer Set 4 8 3 6 7 4 5 7 10

Load 2.0ul on gel

08July2010.jpg

Library preparation by Nextera DNA sample Prep kit

Tagmentation Reaction

Reaction mixture conc. '
AGM 15.6 ng/ul 1.5
Nextera Reaction Buffer LMW 5x 2
Nextera Enzyme mix (Roche Titanium-compatible) ?? 0.5
H2O 6
total 10
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 buffer 2x 12.5
Nextera Primer Cocktail 50x 0.5
Nextera Adaptor1 50x 0.5
Nextera PCR enzyme 2.5 U/ul 0.5
H2O 8.5
total 25
72C 3 min
95C 30 sec
95C 10 sec
55C 30sec
72C 3min
15 cycle

laod 5.0 ul of reaction

0151.jpg

Tagmentation Reaction

Reaction mixture conc. '
AGM 15.6 ng/ul 0.75
Nextera Reaction Buffer LMW 5x 2
Nextera Enzyme mix (Roche Titanium-compatible) ?? 0.5
H2O 6.75
total 10
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 buffer 2x 12.5
Nextera Primer Cocktail 50x 0.5
Nextera Adaptor1 50x 0.5
Nextera PCR enzyme 2.5 U/ul 0.5
H2O 8.5
total 25
72C 3 min
95C 30 sec
95C 10 sec
55C 30sec
72C 3min
18 cycle

laod 5.0 ul of reaction

0153.jpg