User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/30

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Library preparation for Alien genes

Gel extraction

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

load all

extracted at 23 and 24 min (400 ~ 500 bp)

0134.jpg

Library confirmation by PCR

Reaction mixture conc. 1x x4 ' lane Template Primer set
Template (Adapter ligated AGM fragments, purified by MinElute) 0.5 1 2 days ligation (2x buffer) Primer Atitin + Btitin
TopTaq Buffer 10x 1 4 2 2 days ligation (2x buffer) Primer Atitin
dNTP mix 10 mM each 0.2 0.8 3 After gel excision Primer Atitin + Btitin
Primer mix 5 uM each 0.5 4 After gel excision Primer Atitin
TopTaq 5 U/ul 0.05 0.2 5 MilliQ (w/o PCR)
Milli Q 7.75 31
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min

load 4.0 ul of sample

0137.jpg