miniprep was used from GFP bacteria we grew before-

3/25 miniprep was done for plasmid: pIDMv5K-GREEN

Traditional Restriction Digest

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".