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!
