User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/24

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Re-Amp Alien Gene LongPCR

Re-amp Mix 19 and 21

' [CONC] 1X 2 ' ' AMP Lane Template MIX Contents ' Size Fosmid
Water 10.6 21.2 19 CCA3 111 AV10106 FWD AV10108.2 RVS 13400 AV_TEL_L_B
Takara LA Taq 5U 0.2 0.4 21 CCA3 55 AV10108 FWD AV10111 RVS 14846 AV_TEL_L_B
Takara LA Buffer 10X 2 4
dNTP 2.5uM each 3.2 6.4
*Template 1ng 2 4
Primer Mix 5uM each 2
10.0ul 36 18.0ul/RXN
add 2.0 Primer Mix
* CCA3
PCR Cycle
94C 1 min
98C 10 sec
65C 15 min
30X

load 2.0 ul of sample

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Library preparation for Alien genes

Confirmation of adapter ligation by PCR

Reaction mixture conc. 1x x4 ' lane Template Primer set
Template (Adapter ligated AGM fragments, purified by MinElute) 0.25 1 1 2 days ligation (21.9 ng/ul) Primer Atitin + Btitin
TopTaq Buffer 10x 1 4 2 2 days ligation (21.9 ng/ul) Primer Atitin
dNTP mix 10 mM each 0.2 0.8 3 3 days ligation (31.0 ng/ul) Primer Atitin + Btitin
Primer mix 5 uM each 0.5 4 3 days ligation (31.0 ng/ul) Primer Atitin
TopTaq 5 U/ul 0.05 0.2
Milli Q 8 32
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x30
72C 5 min

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