01/14/10
- ✓ Minipreps: KAH117, 118/MV2 (6 each); KAH94 (2), KAH95 (2) retransformations
- ✓ Assemblies: KAH94, 95/JDS47; KAH94, 95/JDS48
- ✓ Re-transform: KAH117, 118/MV2
Minipreps
> Check with E/P digests
| Reagent |
1-12 |
13-16
|
Expected:
1-6. KAH117/MV2 = 7758
7-12. KAH118/MV2 = 7930
13,14. KAH94 = 5011
15,16. KAH95 = 5083
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
5.0 |
2.0
|
| 10X buffer |
1.5 |
1.5
|
| EcoRI |
1.0 |
1.0
|
| PstI |
1.0 |
1.0
|
| dH2O |
6.5 |
9.5
|
| |
15 μL |
15 μL --> 37°C/ ~15 min.
|
--> Re-transform 117, 118/MV2 to isolate clones without mysterious extra bands
Assemblies
- KAH94/JDS47: (1) KAH94/(N, blunted)/5011 + JDS47/(BglII, blunted)/7509 ✓
- KAH95/JDS47: (2) KAH94/(N, blunted)/5083 + "
- KAH94/JDS48: KAH94/(N, blunted)/5011 + JDS48/(BglII, blunted)/7519 ✓
- KAH95/JDS48: KAH94/(N, blunted)/5083 + "
> Digests (Fermentas FD)
| Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
| DNA (plasmid) |
5.0
|
| 10x buffer |
3.0
|
| NotI |
2.0
|
| dH2O |
20.0
|
| |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s of inserts
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH94(N) |
0.085 |
1.80 |
4.3
|
| 2. KAH95(N) |
0.073 |
2.08 |
3.7
|
> 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] = 3.3 ng/μL
- KAH95 (max. vol.) [final] = 2.8 ng/μL
- JDS47 (use max. vol, measure after cleaning)
- JDS48 (use max. vol, measure after cleaning)
> Clean vector DNA using Zymo DNA Clean & Concentrator
| Sample |
OD260 |
260/280 |
ng/μL
|
| 3. JDS47(BglII) |
0.286 |
1.79 |
25.6
|
| 4. JDS48(BglII) |
0.817 |
1.85 |
40.9
|
> Dephosphorylation (Roche)
| Reagent |
Volume
|
| DNA (clean) |
up to 17 μL (400 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
|
| dH2O |
---
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 20 ng/μL
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 01/15/10
|
| 1. KAH94(N, blunted)/5011, 20 ng + JDS47(BglII, blunted, dp)/7509, 15 ng |
KAH94/JDS47 0 colonies (re-do)
|
| 2. KAH95(N, blunted)/5083, 20 ng + " |
KAH95/JDS47 0 colonies (re-do)
|
| 3. JDS47(BglII, blunted, dp)/ 15 ng |
3 colonies
|
| 4. KAH94(N, blunted)/5011, 20 ng + JDS48(BglII, blunted, dp)/7519, 15 ng |
KAH94/JDS48 1 colony (re-do)
|
| 5. KAH95(N, blunted)/5083, 20 ng + " |
KAH95/JDS48 0 colonies (re-do)
|
| 6. JDS48(BglII, blunted, dp)/ 15 ng |
3 colonies
|
| |
1 |
2 |
3 |
4 |
5 |
6
|
| Insert DNA |
6.0 |
6.0 |
--- |
6.0 |
6.0 |
---
|
| Vector DNA |
1.3 |
1.3 |
1.3 |
1.3 |
1.3 |
1.3
|
| 2x lgn buf (Roche) |
8.3 |
8.3 |
8.3 |
8.3 |
8.3 |
8.3
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
--- |
--- |
6.0 |
--- |
--- |
6.0
|
| |
16.4μL |
16.4μL |
16.4μL |
16.4μL |
16.4μL |
16.4μL --> R.T./ 30 min.; Add 30 μL Z-DH5α
|
|
Project name
Main project page
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