Summary
Setting up the ligation (CMV/MV9 & BL01)
- Calculate how many ng of insert you need to get a 4:1 ratio of insert molecules to 50 ng vector molecules
X ng BL01 = (bp BL01 = 2520 / bp CMV/MV9 = 5197) x 4 x 50 ng CMV/MV9 = 96.979ng insert (BL01)
- Calculate how many μL of insert and vector you will need for each ligation:
X μL BL01 = 96.979ng BL01 ÷ (BL01 concentration = 10.694ng/μL) = 9.06854μL X μL vector = 50 ng CMV/MV9 ÷ (CMV/MV9 concentration = 59.108ng/μL) = 0.8459μL
- Set up your ligation reaction(s) in sterile 0.5mL tubes as shown here:
|
Ligation |
Negative Control
|
Insert DNA (X ng) |
9μL |
none
|
Vector DNA (50 ng) |
0.85μL |
same
|
10x Biolabs T4 Ligase Buffer |
2.0 μl |
same
|
Biolabs T4 Ligase |
1.0 μl |
same
|
dH2O |
7.15μL |
16.15μL
|
|
20.0 μL total |
same
|
|
- Mix the reaction(s) thoroughly by flicking the tube.
- Incubate at room temperature overnight.
|