04/18/2013
Building a Reporter Gene to Determine Chromatin Protein Function
- Ligation of KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and cloning in DH5α-T.
- Assembly strategy: The backbone KAH201(S/P)/3403 , the insert is KAH182(X/P)/1675.
Digests
Plasmid DNA |
25.00 μl
|
Enzyme 1* |
1.0 μl
|
Enzyme 2* |
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.
|
- Enzyme 1 for KAH182 is Xba1 and for KAH201 is Spe1.
- Enzyme 2 for KAH182 and KAH201 is Pst1.
- Make the (1%) agarose glee and add 30μl of restricted vector in one well and 10 μl of ladder.
Ligation |
|
1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng |
KAH182/KAH201 2:1 No Colonies
|
2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng |
KAH182/KAH201 3:1 One Colony
|
3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng |
KAH182/KAH201 4:1 Two Colonies
|
4. KAH201(S/P)/size, 36.29ng (Control Plate) |
No Colonies
|
- Calculations are for the ng of insert we need to get a 2:1, 3:1 and 4:1 ratios of insert molecules to 50 ng vector molecules
|
1 (2:1) |
2 (3:1) |
3 (4:1) |
4 (- Ctrl)
|
Insert DNA |
3.11 |
4.66 |
6.21 |
---
|
Vector DNA |
1.37 |
1.37 |
1.37 |
1.37
|
2x lgn buf (Roche) |
5.48 |
7.03 |
8.58 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
0.00 |
0.00 |
0.00 |
2.63
|
|
10.96 μL |
14.06 μL |
17.16μl |
10μL
|
- The incubation time for the ligation process was 15min at room temperature.
- Fast transformation, 15 min on ice.
Confirm The Assembly
Plasmid DNA |
2.0 μl
|
EcoR1 |
1.0 μl
|
Pst1 |
1.0 μl
|
10x FastDigest buffer + green loading dye |
1.5 μl
|
dH2O |
9.5 μl
|
|
15.0 μl total
|
Incubate at 37°C for 10 minutes.
|
- Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.
|