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

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Contents

Library preparation for Alien genes

Gel extraction

lane '
1empty
2Library
3empty
42log ladder (NEB) 3.0 ug
5100 ladder (TaKaRa) 1.0 ug
M1kb plus DNA ladder (Invitrogen) 2.0 ug

load all

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

Image:0134.jpg

Library confirmation by PCR

Reaction mixture conc. 1x x4 ' lane Template Primer set
Template (Adapter ligated AGM fragments, purified by MinElute)0.512 days ligation (2x buffer)Primer Atitin + Btitin
TopTaq Buffer10x1422 days ligation (2x buffer)Primer Atitin
dNTP mix10 mM each0.20.83After gel excisionPrimer Atitin + Btitin
Primer mix5 uM each0.54After gel excisionPrimer Atitin
TopTaq5 U/ul0.050.25MilliQ (w/o PCR)
Milli Q7.7531
10 ul
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min

load 4.0 ul of sample

Image:0137.jpg


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