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

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Contents

Adenita Culture 454 Amplification Adapter extension

EX Takara RXN Mixture conc 1X 34
Milli Q water6.15209.1
Takara EX Buffer10X134
dNTP2.5uM0.827.2
Primer Mix20uM1
EX Taq5U0.051.7
TemplateOleg PCR 1:101
272
8.0ul / RXN add 1.0ul Primer and 1.0ul PCR template
PCR cycle
98C10 sec
60C1 min
x15
Primer Colonies -----> 11 27 29 32 40 41 43 50 54 55 56
DHSP82 F2/R2ok =1ok = 5ok = 7ok = 10ok = 13ok = 16ok = 19? = 22ok = 25ok = 28ok = 31
BHist F1/Hok =2failedok = 8ok = 11ok = 14failedok = 20ok = 23ok = 26ok = 29? = 32
EMyosin F2/R2ok =3failedfailedfailedfailedfailedfailedfailedok = 27failedfailed
FRhook =4ok = 6ok = 9ok = 12ok =15ok = 17ok = 21ok = 24failedok = 30ok = 33
CHistone 2failedfailedfailedfailedok = 18failedfailedfailedfailedfailed
Primer Set113584572678

Loaded 2.0ul

Image:29June10.jpg

Library preparation for Alien genes

Library amplification

Reaction mixture conc. 1x
template (after adapter ligation 1 day liagtion)33.8ng/ul2.5
Atitin primer10uM0.5
Btitin primer10uM0.5
Phusion DNA polymerase2x12.5
MilliQ9
total25
98C30 sec
98C10 sec
65C30 sec
72C30 sec
25 cycle
72C5 min

load 1.0 ul of sample

Image:0133.jpg

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