05/22/15
- Ryan - Bra minipreps & digests
- Cas-tone - minipreps & digests
- Ben - luc replacement donor plasmid
Ryan - Bra minipreps & digests
- Minipreps
- Sigma GenElute kit, elute w/ 75 μL elution sln.
- Digests
- Check with EcoRI digests - promoter insertion generates a new EcoRI site
- Note: BraR = 776, pBra = 214
Reagent
|
Volume
|
Expected: 1-8. pBra-BraR_MRV = 2989, 1202 empty vector = 3977
|
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
dH2O |
10.5
|
|
15 μL
|
--> 37°C/ ~15 min.
Cas-tone - H3_MV11 minipreps & digests
- Cultures
- Pick 2 colonies from each ligation plate
- Make streak library & 5 mL cultures
- Digests
- Check with S/HindIII digests - H3 insertion destroys SpeI site
- Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
Reagent
|
Volume
|
Expected: 1,2. H3-1-40_MV11 = 2345, 7161+## 3,4. H3-1-60_MV11 = 2345, 7162+## 5,6. H3-1-80_MV11 = 2345, 7162+## 7,8. H3-1-116_MV11 = 2345, 7162+## 9,10. H3-1-136_MV11 = 2345, 7162+##
|
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
SpeI |
1.0
|
HindIII |
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
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
|
|