October 19, 2013
Ligation of CMV + MV9
Part
|
Concentration
|
CMV (insert) |
15.535 ng/μL
|
MV9 (vector) |
20.838 ng/μL
|
Calculations
ng insert = bp insert/bp vector * 2 * 50
ng insert = 588/4611 * 2 *50 = 12.75211 ng
μL insert = ng insert/concentration insert
μL insert = 12.75211/15.535 = .82μL
μL vector = 50 ng/concentration vector
μL vector = 50/20.838 = 2.4 μL
|
1
|
Control
|
DNA Insert |
0.8 |
0
|
DNA Vector (MV9) |
2.4 |
2.4
|
T4 Ligase |
1 |
1
|
Lign Buffer (2x) |
5 |
5
|
dH2O |
0.8 |
1.6
|
Total |
10 µL |
10 µL
|
Bacterial Transformation of CMV Promoter + MV9
- Warm 100 μg/mL Amp agar plate at 37°C
- Thaw fresh tube of DH5α Turbo cells on ice
- Add 30 μL of DH5α Turbo Cells to DNA + dH2O.
- This was also done for transformation of part KAH126
- Incubate cells + DNA on ice for 5 minutes.
- Label pre-warmed plate.
- Transfer cells + DNA onto agar.
- Add 8-10 glass beads, shake, then discard beads.
- Incubate plates at 37°C overnight and retrieve next day.
|