Prerequisite Protocols

• None

Materials

• Miniprepped Backbone Plasmid (ideally normalized to 100ng/uL)
• Miniprepped Donor Plasmid or PCR Fragment
• 0.2mL PCR Tubes
• Restriction Enzymes (cut site present on both plasmid backbone and insert fragment)
• 10x Cutsmart Buffer

Equipment

• Vortexer
• PCR Machine
• Ice Bucket
• Pipettes (20uL, 2uL)

Step By Step

1. Preheat a PCR machine to 37C. Thaw a vial of 10x Cutsmart Buffer (New England Biolabs) on ice.
2. To a sterile 0.2mL PCR tube, add 16uL of backbone plasmid. Label the tube with a "V" for "vector".
3. To another sterile 0.2mL PCR tube, add 16uL of donor plasmid which contains the insert you wish to clone. Label this tube with an "I" for "insert".
4. Thaw and dispense 2uL of 10x Cutsmart Buffer (New England Biolabs) to each tube. Keep the Cutsmart Buffer on ice once thawed.
5. To each tube add 1uL of the first restriction enzyme in your cloning plan.
6. To each tube add 1uL of the second restriction enzyme in your cloning plan.