User:Karmella Haynes/Notebook/PcTF Genomics/Entry Base
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mm/dd/yy
- Line item 1
- Line item 2
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 |
|}