11/18/09
- Unfinished line item
- ✓ Finished line item
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
> Digests (Fermentas FD)
--> Specific notes
| 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.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH81 (X/P) |
0.519 |
1.67 |
25.9
|
| 2. huPcCD (E/S) |
1.590 |
1.76 |
79.5
|
| 3. mPcCD (E/S) |
1.604 |
1.76 |
79.5
|
| 4. flyPcCD (E/S) |
0.903 |
1.66 |
45.2
|
| 5. fshPcCD (E/S) |
1.482 |
1.74 |
74.1
|
| 6. hCBX8 (E/S) |
0.691 |
1.74 |
34.5
|
| 7. flyPc (E/S) |
0.118 |
1.44 |
5.9
|
| 2. KAH74 (E/X) |
0.188 |
1.37 |
9.4
|
> 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
|
> Ligations (*Note: use insert + old backbone in calculations)
| Ligation |
Plate results (lig : neg crtl) 11/19/09
|
| 1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng |
KAH86 >10:1 (Pick 2)
|
| 2. KAH48(S/P)/ 20 ng |
|
| 3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng |
KAH87 >10:1 (Pick 2)
|
| 4. mPcCD(E/S)/3386*, > 42 ng + " |
KAH88 >10:1 (Pick 2)
|
| 5. flyPcCD(E/S)/3386*, > 42 ng + " |
KAH89 >10:1 (Pick 2)
|
| 6. fshPcCD(E/S)/3386*, > 42 ng + " |
KAH90 >10:1 (Pick 2)
|
| 7. hCBX8(E/S)/1170, > 42 ng + " |
KAH91 >10:1 (Pick 2)
|
| 8. hCBX8(E/S)/1170, > 42 ng + " |
KAH92 >10:1 (Pick 2)
|
| 9. KAH74(S/P)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9
|
| Insert DNA |
1.4 |
--- |
1.7 |
1.7 |
1.7 |
1.7 |
0.5 |
2.5 |
---
|
| Vector DNA |
0.6 |
0.6 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
---
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.5 |
5.5 |
5.5 |
5.5 |
5.5 |
5.5 |
5.5
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
2.0 |
3.4 |
0.3 |
0.3 |
0.3 |
0.3 |
1.5 |
--- |
2.0
|
| |
10 μL |
10 μL |
11 μL |
11 μL |
11 μL |
11 μL |
11 μL |
11.5 μL |
11 μL
|
Oligo annealing
- NLS myc stop (KAH011)
- K-stop (KAH012)
- Adapter BglII/EcoRI
- Adapter PstI/BglII
| DNA (oligos, 100 μM) |
up to 18 μL
|
| 10x annealing buffer |
2.0
|
| dH2O |
---
|
| |
20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|
|
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