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 Karmella's BioBrick Cloning
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12/11/12
- Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
- Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning
Minipreps
- pSB1A3-RFP (retransformation from 12/10/12)
- Concentrations: (1) 260 = , 260/280 = , ng/μL = ; (2) 260 = , 260/280 = , ng/μL = ;
- Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
| Reagent
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Volume
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Expected:
3. pSB1A3 = size
4. BB2 = size
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15 μL/lane; 1% agarose; Ladder
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| DNA(plasmid) |
10.0 μL
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| 10X buffer |
1.5
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| EcoRI |
1.0
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| PstI |
1.0
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| dH2O |
9.5
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15 μL --> 37°C/ ~15 min.
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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
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Volume
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| DNA (plasmid) |
up to 25 μL
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| 10x buffer |
3.0
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| enzyme 1 |
1.0
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| 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
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| 1. Digested part (a/b) |
--- |
--- |
---
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| 2. Digested part (c/d) |
--- |
--- |
---
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- Dephosphorylation (Roche)
| Reagent
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Volume
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| DNA (clean digest) |
up to 17 μL (500 ng)
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| 10x buffer d.p. |
2.0
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| phosphatase |
1.0
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| dH2O |
---
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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 #)
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| 2. vector(c/d)/ ## ng |
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1 |
2 |
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| Insert DNA |
### |
--- |
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| Vector DNA |
### |
### |
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| 2x lgn buf (Roche) |
### |
### |
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| T4 ligase (NEB) |
1.0 |
1.0 |
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| dH2O |
### |
### |
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# 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.)
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| 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
