4/5/12
Number beside vector and insert is number of base pairs
- 1. flyPCD (3386) + KAH204 (2328)
- 2. flyPCD (3386) + KAH205 (1251)
- 3. flyPCD (3386) + KAH206 (1551)
- 4. flyPCD (3386) + KAH225 (1251)
- 5. flyPCD (3386) only
| '
|
1
|
2
|
3
|
4
|
5
|
| DNA Insert KAH### |
1.3 |
0.4 |
0.5 |
0.3 |
-----------
|
| DNA Vector(flyPCD) |
0.3 |
0.3 |
0.3 |
0.3 |
0.3
|
| T4 Ligase |
1 |
1 |
1 |
1 |
1
|
| Lign Buffer (2x) |
5 |
5 |
5 |
5 |
5
|
| dH2O |
2.4 |
3.3 |
3.2 |
3.4 |
3.6
|
|
10 µL |
10 µL |
10 µL |
10 µL |
10 µL
|
|
|
- In a .5 mL tube, pipette the ligation buffer first.
- Then water, DNA insert, vector, and T4 ligase.
- Once completed, allow them to incubate for 10 minutes.
- Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
- Then follow transformation process:
Transformation Process
- Warm five 100 μg/mL Amp agar plates at 37 °C
- Thaw fresh tube of DH5α Turbo cells on ice
- Include #5 tube, water only, no DNA (negative control)
- Incubate cells + DNA on ice for 5 min.
- Label pre-warmed plates
- Transfer cells + DNA onto agar
- Add 10 - 15 sterile glass beads, shake, discard beads
- Incubate plates at 37 °C overnight
|
 Ligation Results
10:1 ratio with negative control
|
Engineering PC-TFs
