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
- Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.
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
| 
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
|
PCR
- 95°C, 3 min.
- [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 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
|
| 1. pSB1A3 | 0.126 | 1.888 | 77.357
|
| 2. hPCD | 0.069 | 2.192 | 17.025
|
| 3. BL01 | 0.121 | 1.898 | 66.878
|
Dilutions: to final concentration of 20 fmol/μL
| 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
|
Golden Gate Reactions
- hPCD + BL01 + pSB1A3
- pSB1A3
| Reagent | 1 | 2
|
| gg2 pSB1A3 | 1.0 | 1.0
|
| gg3 hPCD | 1.0 | ---
|
| gg4 BL01 | 1.0 | ---
|
| 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
|
Thermal cycling
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 20 min.
- 80°C, 20 min.
- 4°C, ∞
|
Karmella's BioBrick Cloning
Main project page
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