mm/dd/yy
Minipreps
> Check with E/P digests
| Reagent | Volume
| Expected:
1. BB 1 = size
2. BB2 = size
|
|
| DNA(plasmid) | 2.0 μL
|
| 10X buffer | 1.5
|
| EcoRI | 1.0
|
| PstI | 1.0
|
| dH2O | 9.5
|
| | 15 μL --> 37°C/ ~15 min.
|
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
> Digests (Fermentas FD)
--> Specific notes
| Reagent | Volume |
|
|
| DNA (plasmid) | up to 25 μL
|
| 10x buffer | 3.0
|
| enzyme 1 | 1.0
|
| enzyme 2 | 1.0
|
| dH2O | ---
|
| | 30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
| 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
|
| dH2O | ---
|
| | 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
> Ligations
| Ligation | Plate results (lig : neg crtl) mm/dd/yy
|
| 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 |
|
Oligo annealing
- New BB 1
- New BB 2
| DNA (oligos, 100 μM) | up to 18 μL (3 μL ea.)
|
| 10x annealing buffer | 2.0
|
| dH2O | ---
|
| | 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|
|
Karmella's BioBrick Cloning
Main project page
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