06/05/2013
Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)
- Ligation of construct BD001(KAH182-KAH201) and mammalian vector V0200 and cloning in Bl21-DE3.
- KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121.
- Assembly strategy: The backbone V0200(N/S)/4442 , the insert is BD001 (KAH182-KAH201) (N/S)/1901.
Digests
 (1 & 2) BD001 (KAH182-KAH201), 1901 bp, (3 & 4) V0200, 4442 bp, 1, 2, 3 and 4 are cut with Spe1 and Not11
| Plasmid DNA |
15.00 μl
|
| NotI |
1.0 μl
|
| SpeI |
1.0 μl
|
| 10x FastDigest buffer + green loading dye |
3.00 μl
|
| dH2O |
10.00 μl
|
| |
30.0 μl total
|
| Incubate at 37°C for 10 minutes.
|
| Ligation |
|
| 1. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng |
KAH182/KAH201 3:1 Six Colonies
|
| 2. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng |
KAH182/KAH201 4:1 One Colony
|
| 3. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng |
KAH182/KAH201 5:1 One Colony
|
| 4. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng |
KAH182/KAH201 10:1 One Colony
|
| 5. KAH201(S/P)/size, 36.29ng (Control Plate) |
No Colony
|
- Calculations are for the ng of insert we need to get a 3:1, 4:1, 5:1 and 10:1 ratios of insert molecules to 20 ng vector molecules
| Reactions |
1 (3:1) |
2 (4:1) |
3 (5:1) |
4 (10:1) |
5 (- Ctrl)
|
| Insert DNA |
1.22 |
1.63 |
2.04 |
4.08 |
---
|
| Vector DNA |
1.62 |
1.62 |
1.62 |
1.62 |
1.62
|
| 2x lgn buf (Roche) |
5.00 |
5.00 |
5.00 |
5.70 |
5.00
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
1.16 |
0.75 |
0.34 |
--- |
2.38
|
| |
10.00 μL |
10.00 μL |
10.00μl |
12.40μL |
10μL
|
- The incubation time for the ligation process was 15min at room temperature.
- Reaction number 1, 2, 3, 4, 5 40μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 60 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar.
|
PcTF Subcloning in E. coli
