Protocols
This is a protocol for a level 0 digest/ligation reaction.
- Set up a reaction mixture containing:
- 1.5μl 50ng acceptor plasmid (PUDP2)
- 3μl insert DNA
- 1μl T4 ligase
- 2.5μl T4 ligase buffer
- 1μl Esp3I
- 2μl Cutsmart buffer - Place reaction mixtures into a thermocycler and set up a 30X cycle of 37°C for 10 minutes then 16°C for 10 minutes
This is a protocol for a level 1 digest/ligation reaction.
- Set up a reaction mixture containing:
- 100ng acceptor plasmid (PGB-A2)
- Insert DNA in a 3:1 ratio to the acceptor plasmid
- 1μl NEB Golden gate assembly mix
- 1μl NEB Golden gate buffer
- Water to make the reaction up to 20μl
- Place the reactions into a thermocycler and set up a 30X cycle of 37°C for 1 minute then 16°C for 1 minute. With a final 5 minutes at 55°C after the 30 cycles
This is a protocol for transformation of E. coli
- Thaw 50μl aliquots of competent E. coli (DH10α) on ice
- Add 4μl DNA to the cells
- Leave on ice for 10 minutes
- Place cells in a 42°C water bath for 1 minute
- Place cells back on ice for 5 minutes
- Add 1ml of LB broth to the reaction mixtures
- Incubate at 37°C for 1 hour in a shaking incubator
- Spin the cells down in a centrifuge at 13K for 1 minut
- Remove 800μl of supernatant, then resuspend the cell pellet in the remaining 200μl of supernatant
- Plate out the 200μl of resuspended cells and grow overnight at 37°C
This is a protocol for Agrobacterium tumefaciens transformation
- Thaw 50μl aliquots of agrobacteria on ice
- Add 5μl DNA to the bacteria
- Leave on ice for 5 minutes
- Add the reaction mixtures to liquid nitrogen for 5 minutes
- Incubate the reaction mixtures at 37°C for 5 minutes
- Add 1ml of LB broth to the reactions
- Incubate at 28°C in a shaking incubator for 2 hours
- Spin the cells down in a centrifuge at 13K for 1 minute
- Remove 800μl of supernatant, then resuspend the cell pellet in the remaining 200μl of supernatant
- Plate out the 200μl of resuspended cells on agar plates containing Kanamycin and Rifamycin
- Seal the plates with parafilm and incubate at 28°C for 2 days
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