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
|
|
|
|
45
|
|
|
|
9.0ul / RXN add 1.0ul Primer
|
|
|
|
|
|
|
|
|
| PCR cycle |
|
|
|
| 98C |
10 sec |
|
|
| 60C |
1 min |
|
|
| x35 |
|
|
|
|
|
|
|
|
|
|
|
| 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 |
|
|
|
|
|
|
|
|
|
| *Positive Control = AV 20ng |
|
|
|
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 |
|
|
|
|
|
|
|
| 5X Seq Buffer |
5X |
0.4 |
|
|
|
|
|
|
|
| Water |
|
0.6 |
|
|
|
|
|
|
|
| Template * |
~5ng/ul |
4 |
|
|
|
|
|
|
|
|
|
|
2.0ul / RXN |
|
|
|
|
|
|
| *QIAquick Cleaned PCR product |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| Cycle Seq Program |
|
|
|
|
|
|
|
|
|
| 96C |
10 sec |
|
|
|
|
|
|
|
|
| 50C |
5 sec |
|
|
|
|
|
|
|
|
| 60C |
4 min |
|
|
|
|
|
|
|
|
| x60 |
|
|
|
|
|
|
|
|
|
|
Alien genes
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