User:Alexander S Mikheyev/Notebook/bee methylome/2010/04/14

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Library preparation

Library amplification

Reaction mixture conc. Callow Mature
template (after bisulfite conversion) ?? 2.5 5
PCR primer PE 1.0 ?? 0.5 0.5
PCR primer PE 2.0 ?? 0.5 0.5
Phusion DNA polymerase 2x 12.5 12.5
MilliQ 9 6.5
total 25 25
98C 30 sec
98C 10 sec
65C 30 sec
72C 30 sec
25 cycle
72C 5 min

load 1.0ul of reaction

0078.jpg

Gel extraction

lane '
1 Callow
2 empty
3 empty
4 2log ladder (NEB) 3.0 ug
5 100 ladder (TaKaRa) 1.0 ug
6 empty
7 empty
8 Mature
M 1kb plus DNA ladder (Invitrogen) 2.0 ug

load all (E-Gel 2.0%)

0080-1.jpg

Followed by purification using QIAquick PCR purification kit