Continued from 6/4/12
|Insert DNA (X ng)
|Vector DNA (20 ng)
|2x Roche Rapid Ligation buffer
|New England Biolabs T4 ligase
||10.0 μL total
|Mix the reaction(s) thoroughly by flicking the tube.|
Incubate at room temperature for 10 minutes.
Ligate (paste) the DNA fragments together 15 minutes
- Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
X ng insert = (bp insert / bp vector) x 2 x 50 ng vector
- Calculate how many μL of insert and vector you will need for each ligation:
X μL insert = desired ng insert ÷ insert concentration ng/μL (do the same for vector)
- Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown below:
- Note: Because the low concentration of vector we planned to taked 20 ng vectore molecules instead of 50.
Xng inser= (1144 / 3472) x 2 x 20 ng vector=14
Vectore= (20 ng/16μL)= 1.25 μL
Insert= (14ng/8μL)=2 μL
Xng inser= (1144 / 2102) x 2 x 20 ng vector=21
Vectore= (20 ng/17μL)= 1.17 μL
Insert= (21ng/8μL)=2 μL
Transform bacteria with the ligated plasmids 30 minutes
- Warm selection agar plates at 37°C.
- Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per ligation.
- Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
- Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date.
- Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
- Incubate overnight at 37°C to get colonies
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.