User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/30

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454 library preparation (Alien Genes Mix)

Fragmentation

Reaction mixture conc. 1x
Alien Genes mix 368 ng/ul 13.5 ul (5ug)
Fragmentase buffer 10x 10
BSA 100x 1
MilliQ 65.5
sum 90
on ice 5 min
NEB Fragmentase 10 ul
37C 45 min
Purified by QIAquick
Buffer EB 55 ul
3.7 ng/ul total ~200ng

End Repair

Reaction mixture conc. 1x
Alien Genes mix 3.7 ng/ul 54
T4 DNA ligase buffer with 10mM ATP 10x 10
dNTP mix 10mM each 4
T4 DNA ligase 3 U/ul 5
Klenow DNA polymerase 5 U/ul 1
T4 PNK 10 U/ul 5
MilliQ 21
100
20C 30 min
Purified by QIAquick
Buffer EB 40 ul
8.2 ng/ul total ~320ng ??

Adapter ligation

Reaction mixture conc. 1x
Alien Genes mix 8.2 ng/ul 38
T4 DNA ligase buffer with 10mM ATP 10x 5
Adapter_A(5\'Biotinated) 50 uM 1
Adapter_B 50 uM 1
T4 DNA ligase 3 U/ul 5
sum 50
4C O/N