Summary
4/20/13
- 1. fshPCD (vector) + BL01
- 2. fshPCD (vector) + BL05
- 3. flyPCD (vector + BL01
- 4. flyPCD (vector) + BL09
#
|
Biobrick
|
ng/µL
|
1
|
hPCD : mCh : SP1AB (BL01)
|
18.834
|
2
|
fshPCD : mCh : SP1AB (BL05)
|
14.471
|
3
|
flyPCD : mCh : SP1A (BL09)
|
7.302
|
4
|
hPCD : BL01 (BL12)
|
10.84
|
µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)
|
|
1
|
2
|
3
|
4
|
5
|
DNA Insert |
1.4 |
1.8 |
2.0 |
2.5 |
--
|
DNA Vector (Kozak) |
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 |
2.3 |
1.9 |
2.3 |
1.9 |
4.7
|
|
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
- Add 30 μL of DH5α Turbo cells to DNA + dH2O
- Include #5 tube, water only, no DNA (negative control)
- Incubate cells + DNA on ice for 10 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
- No colonies on any of the plates. Used cells that are no longer working.
|