01/03/10
- Assemblies: KAH117-119/MV2; KAH94, 95/ JDS42; KAH94, 95/ JDS43
Assemblies
- KAH117/MV2: (1) KAH117/(E/P)/7758 + MV2/(E/P)/4529 ✓
- KAH118/MV2: (2) KAH118/(E/P)/7930 + "
- KAH119/MV2: (3) KAH119/(E/P)/8002 + "
- KAH94/JDS42: (4) KAH94/(E/P+Ad's)/5011 + JDS42/(BglII)/7468 ✓
- KAH95/JDS42: (5) KAH95/(E/P+Ad's)/5083 + "
- KAH94/JDS43: KAH94/(E/P+Ad's)/5011 + JDS43/(BglII)/7478 ✓
- KAH95/JDS43: KAH95/(E/P+Ad's)/5083 + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
up to 25.0
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
--> For KAH94 & 95: also add old purified E/P digest to melted gel/DNA for column binding.
--> Use 20 μL for final elutions.
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH117(E/P) |
0.089 |
6.67 |
4.5
|
2. KAH118(E/P) |
0.071 |
3.54 |
3.5
|
3. KAH119(E/P) |
0.051 |
4.37 |
2.5
|
4. KAH94(E/P) |
0.204 |
2.22 |
10.2
|
5. KAH95(E/P) |
0.089 |
1.48 |
4.5
|
> Ligations
--> Use dsoligo adapters (Ad BglII/EcoRI and Ad PstI/BglII from 12/28/09) for JDS constructs
Ligation |
Plate results (lig : neg crtl) mm/dd/yy
|
1. KAH117(E/P)/7758, 34 ng + MV2(E/P)/4529, 10 ng |
KAH117/MV2 8 colonies (Pick 4)
|
2. KAH118(E/P)/7930, 35 ng + " |
KAH118/MV2 1 colony (Pick 1)
|
3. KAH119(E/P)/8002, 35 ng + " |
KAH119/MV2 1 colony (Pick 1)
|
4. MV2(E/P)/ 10 ng |
2 colonies
|
5. KAH94(E/P+Ad's)/5011, 40 ng + JDS42(BglII)/7468, 30 ng |
KAH94/JDS42 #:1 (Pick #)
|
6. KAH95(E/P+Ad's)/5083, 41 ng + " |
KAH95/JDS42 #:1 (Pick #)
|
7. JDS42(BglII)/ 30 ng |
|
8. KAH94(E/P+Ad's)/5011, 40 ng + JDS43(BglII)/7478, 30 ng |
KAH94/JDS43 #:1 (Pick #)
|
9. KAH95(E/P+Ad's)/5083, 41 ng + " |
KAH95/JDS43 #:1 (Pick #)
|
10. JDS43(BglII)/ 30 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10
|
Insert DNA |
8.6 |
8.6 |
8.6 |
--- |
7.1 |
7.1 |
--- |
7.1 |
7.1 |
---
|
Vector DNA |
0.4 |
0.4 |
0.4 |
0.4 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5
|
Ad BglII/EcoRI |
--- |
--- |
--- |
--- |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2
|
Ad PstI/BglII |
--- |
--- |
--- |
--- |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
0.2
|
2x lgn buf (Roche) |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
--- |
--- |
--- |
8.6 |
--- |
--- |
7.1 |
--- |
--- |
7.1
|
|
20μL |
20μL |
20μL |
20μL |
20μL |
20μL |
20μL |
20μL |
20μL |
20μL
|
--> R.T./ 30 min.; Add 30 μL Z-DH5α
|