User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/28

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Nuclear Gene Rotifer Culture 454 Amplification

Adenita Culture 13-24

EX Takara RXN Mixture conc 1X 6.5 '
Milli Q water 5.15 33.475
Takara EX Buffer 10X 1 6.5
dNTP 2.5uM 0.8 5.2
Primer Mix 20uM 1
EX Taq 5U 0.05 0.325
Template Instagene 2 13
58.5 9.0ul / RXN
A PCR cycle
98C 10 sec
60C 1 min
x35
Plate 2
Source Plate Well EX ID
Ithaca 1 A2 13
Ithaca 1 A3 14
Ithaca 1 A5 15
Ithaca 1 A6 16
Ithaca 1 B1 17
Ithaca 1 B3 18
Ithaca 1 B5 19
Ithaca 1 C1 20
Ithaca 1 D1 21
Ithaca 1 D3 22
Ithaca 1 D4 23
Ithaca 1 D6 24

Shearing by NEB fragmentase

Reaction mixture conc. 1x (ul) x4
DNA (2ug) 363 ng/ul 5.5 22
Fragmentase buffer 10x 2 8
BSA 100x 0.2 0.8
MilliQ 10.3 41.2
on ice 5 min
Fragmentase 2
37C No. 1 10 min
No. 2 15 min
No. 3 20 min
No. 4 25 min

File:2100 expert High Sensitivity DNA Assay DE72901887 2010-04-28.pdf