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

From OpenWetWare

Jump to: navigation, search
Alien genes Main project page
Previous entry      Next entry

Contents

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 water6.1518.45
Takara EX Buffer10X13
dNTP2.5uM0.82.4
Primer Mix5uM1
EX Taq5U0.050.15
TemplateChelex Extraction1
24
8.0ul / RXN add 1.0ul Primer and 1.0ul Chelex Extraction
Well Culture
1S. pombe
2S. cerevisiae
3negative control

Load 2.0ul sample

Image:01July10.jpg

Library preparation for Alien genes

Library confirmation by PCR

Reaction mixture conc. 1x x3 ' lane Primer set
Template0.250.751Primer Atitin + Btitin
TopTaq Buffer10x132Primer Atitin
dNTP mix10 mM each0.20.63AV10027
Primer mix (AV10027)5 uM each0.5
TopTaq5 U/ul0.050.15
Milli Q824
10 ul
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min

load 5.0 ul of sample

Image:0140-2.jpg

Reaction mixture conc. 1x x2 ' lane Primer set
Template (Adapter ligated AGM fragments)2.5 ng/ul0.511Primer Atitin + Btitin
TopTaq Buffer10x122AV10027
dNTP mix10 mM each0.20.4
Primer mix5 uM each0.5
TopTaq5 U/ul0.050.1
Milli Q7.7515.5
10 ul
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min

load 5.0 ul of sample

Image:0141.jpg


Control experiment (End Repair)

Reaction mixture conc. 1x
10kb mix (gene rich region, fragmented by fragmentase, 500 - 1,000bp)15 ng/ul18.75
T4 DNA lligase buffer with 10 mM ATP10x2.5
dNTP mix10 mM each1
T4 DNA polymerase3 U/ul1.25
Klenow DNA polymerase5 U/ul0.25
T4 PNK10 U/ul1.25
total25
20 C30 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/ul3
Atitin + adapter50 nM0.2
Btitin + adapter50 nM0.2
T4 DNA lligase buffer with 10 mM ATP10x1
T4 DNA ligase400 U/ul1
MilliQ4.6
total10
4 Co/n
Personal tools