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 Karmella's BioBrick Cloning
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01/06/13
- Gibson Assembly: pick colonies & check via colony PCR
- Media: Make LB agar +amp
- Gibson Assembly: redo transformation with more DNA
Gibson Assembly results
- hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
- hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
- hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
- hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
- Pick and streak colonies from plates 3, 5, 7
- Use same pipette tip for colony PCR
| Reagent
|
Volume
|
5x mix
|
| Colony |
--- |
---
|
| 10 μM primer BL9 |
1.0 |
5.0
|
| 10 μM primer BL10 |
1.0 |
5.0
|
| 2x GoTaq |
12.5 |
62.5
|
| dH2O |
10.5 |
52.5
|
| |
25.0 μL |
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PCR
- 95°C, 3 min.
- [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
- 72°C, 3 min.
- 4°C, ∞
Assemblies
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
| Reagent
|
Volume
|
|
| DNA (plasmid) |
up to 25 μL
|
| 10x buffer |
3.0
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
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| dH2O |
---
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| |
30 μL --> 37°C/ ~30 min.
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| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. Digested part (a/b) |
--- |
--- |
---
|
| 2. Digested part (c/d) |
--- |
--- |
---
|
- Dephosphorylation (Roche)
| Reagent
|
Volume
|
| DNA (clean digest) |
up to 17 μL (500 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
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| dH2O |
---
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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| Ligation |
Plate results (lig : neg crtl) mm/dd/yy
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng |
new BioBrick #:1 (Pick #)
|
| 2. vector(c/d)/ ## ng |
|
| |
1 |
2 |
|
| Insert DNA |
### |
--- |
|
| Vector DNA |
### |
### |
|
| 2x lgn buf (Roche) |
### |
### |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
|
| dH2O |
### |
### |
|
| |
# μL |
# μL |
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Oligo annealing
- New BB 1
- New BB 2
| DNA (oligos, 100 μM) |
up to 18 μL (3 μL ea.)
|
| 10x annealing buffer |
2.0
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| dH2O |
---
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20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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Karmella's BioBrick Cloning
