User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/01

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ITS amplification of Yeast culture

Chelex Extraction from Yeast Culture

1) Gently swirl growing culture. Pipette 50.0ul into tube. 2) Spin down at 14,000 rpm. Remove supernatant. 3) Add 200.0ul of Millipore water and pipette mix. 4) Spin down at 14,000 rpm. Remove supernatant. 5) Add 100.0ul Chelex. 6) Boil at 100.0C for 10min. 27C for 1 min. 7) Use 1.0ul for ITS amplification.


EX Takara RXN Mixture conc 1X 3
Milli Q water 6.15 18.45
Takara EX Buffer 10X 1 3
dNTP 2.5uM 0.8 2.4
Primer Mix 5uM 1
EX Taq 5U 0.05 0.15
Template Chelex Extraction 1
24
8.0ul / RXN add 1.0ul Primer and 1.0ul Chelex Extraction
Well Culture
1 S. pombe
2 S. cerevisiae
3 negative control

Load 2.0ul sample

01July10.jpg

Library preparation for Alien genes

Library confirmation by PCR

Reaction mixture conc. 1x x3 ' lane Primer set
Template 0.25 0.75 1 Primer Atitin + Btitin
TopTaq Buffer 10x 1 3 2 Primer Atitin
dNTP mix 10 mM each 0.2 0.6 3 AV10027
Primer mix (AV10027) 5 uM each 0.5
TopTaq 5 U/ul 0.05 0.15
Milli Q 8 24
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min

load 5.0 ul of sample

0140-2.jpg

Reaction mixture conc. 1x x2 ' lane Primer set
Template (Adapter ligated AGM fragments) 2.5 ng/ul 0.5 1 1 Primer Atitin + Btitin
TopTaq Buffer 10x 1 2 2 AV10027
dNTP mix 10 mM each 0.2 0.4
Primer mix 5 uM each 0.5
TopTaq 5 U/ul 0.05 0.1
Milli Q 7.75 15.5
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min

load 5.0 ul of sample

0141.jpg


Control experiment (End Repair)

Reaction mixture conc. 1x
10kb mix (gene rich region, fragmented by fragmentase, 500 - 1,000bp) 15 ng/ul 18.75
T4 DNA lligase buffer with 10 mM ATP 10x 2.5
dNTP mix 10 mM each 1
T4 DNA polymerase 3 U/ul 1.25
Klenow DNA polymerase 5 U/ul 0.25
T4 PNK 10 U/ul 1.25
total 25
20 C 30 min
prurified by MinElute kit
12 ul of buffer EB
16 ng /ul

Control experiment (ligation)

Reaction mixture conc. 1x
10 kb mix (gene rich region, end repaired) 16 ng/ul 3
Atitin + adapter 50 nM 0.2
Btitin + adapter 50 nM 0.2
T4 DNA lligase buffer with 10 mM ATP 10x 1
T4 DNA ligase 400 U/ul 1
MilliQ 4.6
total 10
4 C o/n