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Karmella's BioBrick Cloning
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01/09/13
- Golden Gate assembly: hPCD + BL01 + pSB1A3
- Violacein: plates from Tabor; incubate o/n
Golden Gate Assembly
- Use purified, measured PCR products from 1/05/13
- Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
DNA
|
length (bp)
|
ng/μL
|
Vol.
|
+ dH2O
|
gg2 pSB1A3 |
2000 |
67.7 |
7.7 |
12.3
|
gg3 hPCD |
186 |
22.5 |
2.1 |
17.8
|
gg4 BL01 |
2520 |
55.9 |
11.7 |
8.3
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- hPCD + BL01 + pSB1A3
- pSB1A3
Reagent |
1 |
2
|
gg2 pSB1A3 |
1.0 |
1.0
|
gg3 hPCD |
1.0 |
---
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gg4 BL01 |
1.0 |
---
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10x ligase buffer |
1.0 |
1.0
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NEB T4 lgase |
0.25 |
0.25
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BsmBI |
0.5 |
0.5
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dH2O |
5.25 |
7.25
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|
10.0 |
10.0
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Thermal cycler
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 20 min.
- 80°C, 20 min.
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