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 Karmella's BioBrick Cloning
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01/15/13
- Golden gate assembly: test different ligase buffers
Golden Gate Assembly optimization
| Buffer |
Tris-HCl |
MgCl2 |
DTT |
ATP
|
| 10x Promega |
300 mM (pH 7.8) |
100 mM |
100 mM |
10 mM
|
| 10x NEB |
500 mM (pH 7.5) |
100 mM |
100 mM |
10 mM
|
| 2x Roche |
? |
? |
? |
? |
?
|
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
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
| 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
|
| 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
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Karmella's BioBrick Cloning
