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 Karmella's BioBrick Cloning
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01/10/13
- Violacein: successful growth; re-streak
- Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
- Golden gate trial 2: new PCR primers
Violacein
- Plate 1: Kan plasmid
- Plate 2: Amp plasmid (violacein exp.)
Golden gate trial 1
- Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
Golden gate trial 2
- Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
| Reagent
|
Volume
|
Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
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10 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
0.2 μL
|
| primer 1 |
1.0
|
| primer 2 |
1.0
|
| 2x GoTaq |
25.0
|
| dH2O |
22.8
|
| |
50.0 μL
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PCR
- 95°C, 3 min.
- [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Purification
- Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
- Measure [DNA]
| Sample
|
OD 260
|
260/280
|
ng/μL
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| 1. pSB1A3 |
0.126 |
1.888 |
77.357
|
| 2. hPCD |
0.069 |
2.192 |
17.025
|
| 3. BL01 |
0.121 |
1.898 |
66.878
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Dilutions
| DNA
|
length (bp)
|
ng/μL
|
Vol.
|
+ dH2O
|
| pSB1A3 |
2000 |
77.4 |
6.7 |
13.3
|
| hPCD |
186 |
17.0 |
2.8 |
17.2
|
| BL01 |
2520 |
66.9 |
9.8 |
10.2
|
- hPCD + BL01 + pSB1A3
- pSB1A3
| Reagent |
1 |
2
|
| gg2 pSB1A3 |
1.0 |
1.0
|
| gg3 hPCD |
1.0 |
---
|
| gg4 BL01 |
1.0 |
---
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| 10x ligase buffer |
1.0 |
1.0
|
| NEB T4 lgase |
0.25 |
0.25
|
| BsmBI |
0.5 |
0.5
|
| dH2O |
5.25 |
7.25
|
| |
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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Karmella's BioBrick Cloning
