Calculations resulted in the following:
|| Negative Control
| Insert DNA (BL01, BL05, BL09 respectively)
| Vector DNA (50 ng)
| 10x Biolabs T4 Ligase Buffer
|| 2.0 μl
| Biolabs T4 Ligase
|| 1.0 μl
|| 20.0 μL total
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 45 minutes.
Using the set up ligation products from above.
- DH5α-T cells were thawed in ice.
- Six 1.5mL tubes were setup and labeled; 1 (BL01), 5 (BL05), 9 (BL09), CMV9 (CMV/MV9, ligation benchmark), rhlI (pos control), Neg (negative control).
- 60μL of thawed cells were mixed and then transferred into each of these six empty tubes.
- The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
- The RhlI plasmid was used as a positive control; 1μL of this plasmid was added to 19μL dH2O to equal the other two products' 20μL total volume. Added to the fifth tube with 60μL thawed cells.
- All tubes incubated on ice for 35 minutes.
- All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
- 900μL LB broth (no antibiotic) was pipetted into each of the three tubes.
- Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
- Incubated on shaker for 1 hour at 37°C and 240rpm.
- Centrifuged for 1.5 minutes at 8 x g.
- 500μL media removed from each tube.
- Resuspended cell pellet in remaining 400μL media.
- 300μL cells transferred onto respective plate.
- Sterile glass beads used to spread cells onto plate.
- Placed in the incubator for overnight growth.