User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/06

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Nuclear Gene HS Takara PCR Optimization

HS Takara RXN Mixture 3.0ul Instagene conc 1X 5.5 '
Milli Q water 3.1 17.05
Takara HS Buffer 10X 2 11
dNTP 2.5uM 0.8 4.4
Primer Mix 20uM 1
HS Taq 5U 0.1 0.55
Template Instagene 3 16.5
49.5 9.0ul / RXN
add 1.0ul Primer
HS Takara RXN Mixture 4.0ul Instagene conc 1X 5.5
Milli Q water 2.1 11.55
Takara HS Buffer 10X 2 11
dNTP 2.5uM 0.8 4.4
Primer Mix 20uM 4
HS Taq 5U 0.1 0.55
Template Instagene 4 22
49.5 9.0ul / RXN
add 1.0ul Primer
A PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Primer Amt of Template
1 3 Instagene A2 3
2 3 Instagene A3 3
3 3 Instagene A4 3
4 3 Instagene A5 3
5 3 Instagene A6 3
6 4 Instagene A2 3
7 4 Instagene A3 3
8 4 Instagene A4 3
9 4 Instagene A5 3
10 4 Instagene A6 3
11 3 Instagene A2 4
12 3 Instagene A3 4
13 3 Instagene A4 4
14 3 Instagene A5 4
15 3 Instagene A6 4
16 4 Instagene A2 4
17 4 Instagene A3 4
18 4 Instagene A4 4
19 4 Instagene A5 4
20 4 Instagene A6 4
21 AV 2ng A2 1
22 AV 2ng A3 1
23 AV 2ng A4 1
24 AV 2ng A5 1
25 AV 2ng A6 1
*AV 1ng Positive Control

Load 2.0ul Sample

06May10.jpg


AV 1ng control failed - bad DNA dilution

Dye Dilution Test

1:10 Dye Stock : MilliQ Water

From 06May10 PCR - samples 1-10 Dye test.jpg