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PCR'd the plasmids cut on 12.05.29 to PCR out the individual genes with the correct overhangs (Silver Standard RFC[23] compatible with no frame shift).
Plasmids from 12.05.29:
| Lane |
Part Number |
Gene(s)
|
| 1 |
K118014 |
RBS + crtE
|
| 2 |
K274100 |
RBS + crtE + RBS + crtI + RBS + crtB
|
| 3 |
K274200 |
RBS + crtE + RBS + crtB + RBS + crtI +RBS + crtY
|
| 4 |
K118002 |
crtB
|
| 5 |
K118006 |
RBS + crtB
|
| 6 |
I742161 |
RBS + crtI + RBS + crtB
|
| 7 |
K118003 |
crtI
|
| 8 |
K118005 |
RBS + crtI
|
| Lane |
Gene |
Plasmid |
Primer F |
Primer R
|
| 1 |
crtE |
1 |
P001 |
P002
|
| 2 |
crtE |
2 |
P001 |
P002
|
| 3 |
crtE |
3 |
P001 |
P002
|
| 4 |
crtB |
2 |
P003 |
P004
|
| 5 |
crtB |
3 |
P003 |
P004
|
| 6 |
crtB |
4 |
P003 |
P004
|
| 7 |
crtB |
5 |
P003 |
P004
|
| 8 |
crtI |
2 |
P005 |
P006
|
| 9 |
crtI |
3 |
P005 |
P006
|
| 10 |
crtI |
7 |
P005 |
P006
|
| 11 |
crtI |
8 |
P005 |
P006
|
PCR reaction
| Reagent |
Volume
|
| DNA (plasmid) |
1.0
|
| 10 μM primers |
1.0
|
| 2x GoTaq |
25.0
|
| dH2O |
23.0
|
--> Wang Lab PCR machine, block A
- 95°C/ 3 min.
- [95°C/ 30 sec; 50°C/ 30 sec; 72 °C/ 1 min.] x30
- 72 °C/ 3 min.
- 4 °C/ ∞
> Qiagen PCR cleanup kit; elute w/ 50ul EB2O
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