HCO/LCO Adenita Culture Re-Amp PCR Purification with QIAquick
For failed sequencing samples 8, 12, G
http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx#Tabs=t2
- see page 19.
- 16ul PCR product left for cleaning. Resuspended in 50ul EB buffer.
| Sample ID
|
Nucleic Acid Conc.
|
Unit
|
A260
|
A280
|
260/280
|
260/230
|
Sample Type
|
Factor
|
| 8 |
5.9 |
ng/µl |
0.117 |
0.036 |
3.22 |
2.62 |
DNA |
50
|
| 12 |
5.7 |
ng/µl |
0.114 |
0.064 |
1.77 |
1.93 |
DNA |
50
|
| G |
3.7 |
ng/µl |
0.073 |
0.029 |
2.55 |
19.45 |
DNA |
50
|
Use 4.0ul = 15-20ng for Cycle Sequencing
HCO/LCO Adenita Culture Cycle Sequencing
For failed sequencing samples 8, 12, G
| AMP Lane
|
Template
|
'
|
Sample Number
|
Primer
|
| 1 |
E. Windsor |
B2 |
8 |
HCO
|
| 2 |
Ithaca 1 |
A1 |
12 |
HCO
|
| 3 |
Woods Hole 1 |
C4 |
G |
HCO
|
| 4 |
E. Windsor |
B2 |
8 |
LCO
|
| 5 |
Ithaca 1 |
A1 |
12 |
LCO
|
| 6 |
Woods Hole 1 |
C4 |
G |
LCO
|
| CS Reaction mixture
|
conc.
|
1x
|
3
|
'
|
| Big Dye Terminator |
|
0.5 |
1.5 |
|
| Primer (FWD or RVS) |
5uM |
0.4 |
1.2 |
|
| DMSO |
|
0.1 |
0.3 |
|
| 5X Seq Buffer |
5X |
0.4 |
1.2 |
|
| Water |
|
0.6 |
1.8 |
|
| Template * |
~5ng/ul |
4 |
|
|
|
|
|
6 |
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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