Date
Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)
- Ligation of construct KAH182-KAH201 and mammalian vector V0200 and cloning in DH5α-T.
- KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121.
- Assembly strategy: The backbone V0200(N/S)/4442 , the insert is KAH182-KAH201(N/S)/1901.
Digests
Plasmid DNA |
25.00 μl
|
NotI |
1.0 μl
|
SpeI |
1.0 μl
|
10x FastDigest buffer + green loading dye |
3.00 μl
|
dH2O |
0.00 μl
|
|
30.0 μl total
|
Incubate at 37°C for 10 minutes.
|
Ligation |
|
1. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng |
KAH182/KAH201 3:1 No Colonies
|
2. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng |
KAH182/KAH201 4:1 One Colony
|
3. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng |
KAH182/KAH201 4:1 one Colony
|
4. KAH201(S/P)/size, 36.29ng (Control Plate) |
One Colonies and lots of satellite colonies in the middle of the pate
|
- Calculations are for the ng of insert we need to get a 3:1, 4:1 and 5:1 ratios of insert molecules to 20 ng vector molecules
|
1 (3:1) |
2 (4:1) |
3 (5:1) |
4 (- Ctrl)
|
Insert DNA |
2.85 |
3.80 |
4.76 |
---
|
Vector DNA |
1.00 |
1.00 |
1.00 |
1.00
|
2x lgn buf (Roche) |
5.00 |
5.80 |
6.76 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
0.15 |
0.00 |
0.00 |
3.00
|
|
10.00 μL |
11.60 μL |
13.52μl |
10μL
|
- The incubation time for the ligation process was 15min at room temperature.
- Fast transformation, 15 min on ice.
|