Week 2 Lab: Restriction Digests

Protocol

Part 0: Design

Note: Ignore EcoRI lane: for visualization Note: We are using DNA from mycobacteriophage Kampy

Part 1a: Gel prep

Setting up a 1% agarose gel:

• 0.5 g agarose to plastic flask
• 50 ml 1x TAE to agarose in flask
• Microwave the mixture 1:30 / until agarose dissolves
• Sideways gel tray in box, pour hot agarose/TAE, add comb
• Let set 30 min

Part 1b: Restriction Digest

Chemicals

• 1X Lambda DNA
• 1 uL of each enzyme: EcoRI-HF, HindIII-HF, BamHI-HF, KpnI-HF, EcoRV-HF, SacI-HF, SalI-HF
• Nuclease-free water Equipment and Consumables
• -20ºC freezer
• Incubator (or use a thermocycler or heat block
• “PCR tube rack” (pipette tip holder)

Digest requirements

• 3 ul 0.5 ug/uL DNA – final conc 1.5 ug/20 ul total
• 2 uL 10x buffer
• 1 uL 20 units/uL enzyme – final conc 15 units
• NFW: enough to make 20 uL total mixture

our DNA = 324 ng/uL = 0.324 ug/uL– to get 1.5 ug, we need 4.62 uL of DNA

Plan for Mixtures

• 4.62 uL DNA

• 2 ul 10x buffer

• BstXI: to get final amt 15 ug – 37 – H buffer

  • 1.5 ul of 10 u/ul
  • 11.88 uL NFW

• KpnI: to get final amt 15 ug – 37 – MC buffer

  • use 1.25 ul of 12 u/ul
  • 12.13 uL

• SfiI: to get final amt 15 ug – 50 – B buffer

  • use 1.5 ul of 10 u/ul
  • 11.88 uL NFW

Incubate at temp – 1 hr

Notes/Scratch work

Done: NFW, DNA, Buffer, Enzyme

Running

• BstXI – + assoc buffer – 37oC

  • stock buffer: promega 10x
  • enzyme: 10 u / ul

• BstXI – + assoc buffer – 37oC

• KpnI – + assoc buffer – 37oC

  • stock buffer: promega 10x buffer
  • enzyme: 12 u / ul

• SfiI – + assoc buffer – 50oC

  • stock buffer: promega 10x
  • enzyme: 10 u / ul

See chart on site for reagent concs and quantities

Part 2: Gel Run

• pour TAE over fill line
• load 12 ul ladder
• add 4 ul 6x dye to ea. 20 ul digest
• load 10 ul of the above on the gel
• set up electrophoresis and run ~30 min
• let stain in ethidium bromide 20 min
• image with gel imager

Results