Week 2 Lab: DNA Gel Art

Design

Lane 4 will be left empty. EcoRI is listed here for visual purposes.

Restriction Digest Protocol

Digest components

• X uL NFW to 20 uL
• 2 uL 10X Promega buffer
• 1.5 ug DNA
• 15 units restriction enzyme

Calculations for 10 uL reactions

• Kampy DNA concentration: 324 ng/uL
  • Use 4.6 uL DNA

BstXI

• Stock BstXI concentration: 10 u/uL
  • 1.5 uL enzyme
  • 11.9 uL NFW
• Buffer: Buffer H
• Temperature: 37C

SfiI

• Stock SfiI concentration: 10 u/uL
  • 1.5 uL enzyme
  • 11.9 uL NFW
• Buffer: Buffer B
• Temperature: 50C

KpnI

• Stock KpnI concentration: 12 u/uL
  • 1.25 uL enzyme
  • 12.15 uL NFW
• Buffer: Multicore buffer
• Temperature: 37C

Gel Protocol

Gel components

• 0.50 g agarose
• 50 mL TAE buffer

Running the Gel

• Sample order: ladder, BstXI, KpnI, BstXI, empty, BstXI, SfiI, BstXI
• Add 4 uL 6X loading dye to each restriction digest
• Run 12 uL digest in each well for 40 min at 150V
• Stain the gel in 100 mL Millipore water and 3 drops of ethidium bromide

Results: Phage!