User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/29

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Adenita Culture 454 Amplification Adapter extension

EX Takara RXN Mixture conc 1X 34
Milli Q water 6.15 209.1
Takara EX Buffer 10X 1 34
dNTP 2.5uM 0.8 27.2
Primer Mix 20uM 1
EX Taq 5U 0.05 1.7
Template Oleg PCR 1:10 1
272
8.0ul / RXN add 1.0ul Primer and 1.0ul PCR template
PCR cycle
98C 10 sec
60C 1 min
x15
Primer Colonies -----> 11 27 29 32 40 41 43 50 54 55 56
D HSP82 F2/R2 ok =1 ok = 5 ok = 7 ok = 10 ok = 13 ok = 16 ok = 19 ? = 22 ok = 25 ok = 28 ok = 31
B Hist F1/H ok =2 failed ok = 8 ok = 11 ok = 14 failed ok = 20 ok = 23 ok = 26 ok = 29 ? = 32
E Myosin F2/R2 ok =3 failed failed failed failed failed failed failed ok = 27 failed failed
F Rho ok =4 ok = 6 ok = 9 ok = 12 ok =15 ok = 17 ok = 21 ok = 24 failed ok = 30 ok = 33
C Histone 2 failed failed failed failed ok = 18 failed failed failed failed failed
Primer Set 11 3 5 8 4 5 7 2 6 7 8

Loaded 2.0ul

29June10.jpg

Library preparation for Alien genes

Library amplification

Reaction mixture conc. 1x
template (after adapter ligation 1 day liagtion) 33.8ng/ul 2.5
Atitin primer 10uM 0.5
Btitin primer 10uM 0.5
Phusion DNA polymerase 2x 12.5
MilliQ 9
total 25
98C 30 sec
98C 10 sec
65C 30 sec
72C 30 sec
25 cycle
72C 5 min

load 1.0 ul of sample

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