01/16/10
- ✓ Minipreps: KAH117, 118/MV2 (re-transformation clones, 2 each)
- ✓ Cultures: KAH137(3), 138(3)
- ✓ Assemblies: KAH94, 95/ JDS47; KAH94, 95/ JDS48 (re-do)
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: MV2 = 4529 1,2. KAH117/MV2 = 7758 3,4. KAH118/MV2 = 7930
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
--> KAH117/MV2 worked. Pick 4 more colonies from KAH118/MV2 re-transformation plate
Assemblies
--> Re-do (see 1/14), use higher conc. of insert
--> Increase ligation time (1 hr.)
- KAH94/JDS47: (1) KAH94/(N, blunted)/5011 + JDS47/(BglII, blunted, dp)/7509 ✓
- KAH95/JDS47: (2) KAH94/(N, blunted)/5083 + "
- KAH94/JDS48: KAH94/(N, blunted)/5011 + JDS48/(BglII, blunted, dp)/7519 ✓
- KAH95/JDS48: KAH94/(N, blunted)/5083 + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
10.0
|
10x buffer |
3.0
|
NotI |
2.0
|
dH2O |
15.0
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s of inserts
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH94(N) |
0.360 |
2.57 |
18.0
|
2. KAH95(N) |
0.363 |
2.01 |
18.2
|
> Blunting (NEB Quick Blunting kit)
--> Blunt all DNA (vectors and inserts)
Reagent |
Volume
|
DNA (clean digest) |
up to 19 μL
|
10x blunting buffer |
2.5
|
1 mM dNTP mix |
2.5
|
Blunt Enzyme mix |
1.0
|
dH2O |
---
|
|
25 μL --> R.T./ 15 min.; 70°C/ 10 min.
|
- KAH94 (max. vol.) [final] = 13.7 ng/μL
- KAH95 (max. vol.) [final] = 13.7 ng/μL
> Ligations
Ligation |
Plate results (lig : neg crtl) 01/17/10
|
1. KAH94(N, blunted)/5011, 40 ng + JDS47(BglII, blunted, dp)/7509, 30 ng |
KAH94/JDS47 10:1 (Pick 6)
|
2. KAH95(N, blunted)/5083, 40 ng + " |
KAH95/JDS47 10:1 (Pick 6)
|
3. JDS47(BglII, blunted, dp)/ 30 ng |
|
4. KAH94(N, blunted)/5011, 40 ng + JDS48(BglII, blunted, dp)/7519, 30 ng |
KAH94/JDS48 10:1 (Pick 6)
|
5. KAH95(N, blunted)/5083, 40 ng + " |
KAH95/JDS48 10:1 (Pick 6)
|
6. JDS48(BglII, blunted, dp)/ 15 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6
|
Insert DNA |
3.7 |
3.7 |
--- |
3.7 |
3.7 |
---
|
Vector DNA |
1.3 |
1.3 |
1.3 |
1.3 |
1.3 |
1.3
|
2x lgn buf (Roche) |
6.0 |
6.0 |
6.0 |
6.0 |
6.0 |
6.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
--- |
--- |
3.7 |
--- |
--- |
3.7
|
|
12μL |
12μL |
12μL |
12μL |
12μL |
12μL --> R.T./ 1 hr; Add 30 μL Z-DH5α
|
|