Nuclear Gene Adenita Cultures 454 Amplification
A. ricciae
EX Takara RXN Mixture
|
conc
|
1X
|
5
|
Milli Q water |
|
4.15 |
20.75
|
Takara EX Buffer |
10X |
1 |
5
|
dNTP |
2.5uM |
0.8 |
4
|
Primer Mix |
20uM |
1 |
|
EX Taq |
5U |
0.05 |
0.25
|
Template |
Instagene |
3 |
15
|
|
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|
45
|
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9.0ul / RXN add 1.0ul Primer
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PCR cycle |
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98C |
10 sec |
|
|
60C |
1 min |
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x35 |
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AMP Lane |
Template |
Primer |
|
1 |
A. Ricciae |
B3 |
|
2 |
A. Ricciae |
C3 |
|
3 |
A. Ricciae |
D3 |
|
4 |
A. Ricciae |
E3 |
|
5 |
A. Ricciae |
F3 |
|
6 |
Neg Control |
B3 |
|
7 |
Neg Control |
C3 |
|
8 |
Neg Control |
D3 |
|
9 |
Neg Control |
E3 |
|
10 |
Neg Control |
F3 |
|
11 |
Pos Control |
B3 |
|
12 |
Pos Control |
C3 |
|
13 |
Pos Control |
D3 |
|
14 |
Pos Control |
E3 |
|
15 |
Pos Control |
F3 |
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*Positive Control = AV 20ng |
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Loaded 2.0ul
HCO/LCO Adenita Culture Cycle Sequencing
For failed sequencing sample 14
CS Reaction mixture
|
conc.
|
1x
|
'
|
'
|
AMP Lane
|
Template
|
'
|
Sample Number
|
Primer
|
Big Dye Terminator |
|
0.5 |
|
|
1 |
Ithaca 1 |
A3 |
14 |
HCO
|
Primer (FWD or RVS) |
5uM |
0.4 |
|
|
2 |
Ithaca 1 |
A3 |
14 |
LCO
|
DMSO |
|
0.1 |
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5X Seq Buffer |
5X |
0.4 |
|
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Water |
|
0.6 |
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Template * |
~5ng/ul |
4 |
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2.0ul / RXN |
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*QIAquick Cleaned PCR product |
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Cycle Seq Program |
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96C |
10 sec |
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50C |
5 sec |
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60C |
4 min |
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x60 |
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