Griffitts:In-gel ligation

Materials

• Gel tray
• 1X TAE buffer
• 8-well comb
• autoclave tape
• agarose (for low-melt DNA gels)
• 1 mg/mL ethidium bromide
• 8X loading dye
• microcentrifuge tubes (1 per reaction)
• razor blade
• paper towel
• T4 ligase (on ice)
• 10X T4 ligase buffer
• sterile ddH₂0

Procedure

Gel preparation

• Carefully tape both ends of the tray with autoclave tape
• Insert the 8-well comb
• Start with 50 mL 1X TAE buffer
  • Use the flask labeled "Agarose—keep in gel area"
• Add 0.5 g agarose
  • Use the agarose labeled "for low-melt DNA gels"
• Microwave until dissolved
  • This will take less than 1 minute and will require frequent stirring to prevent a boil-over
• Add 8 μL of 1 mg/mL ethidium bromide and stir
  • This is kept in the fridge
• Allow to cool ~5 minutes
• Pour into the tray
• Place in a level place in the refrigerator and allow agarose to solidify for at least 30 minutes
• Remove tape and comb
  • Remove the comb slowly so you don't tear the wells

Sample preparation

• Add 1/5 volume 8X loading dye to each of your samples

Electrophoresis

• Check to make sure there is enough 1X TAE buffer in the gel box
  • Dr. Griffitts has drawn a black line on the side indicating where is sufficient
  • If you suspect the 1X TAE buffer is old, replace it; you may dump the old 1X TAE buffer down the drain with water
• Place your gel (still on the tray) in the box with the wells away from you
  • If your gel is pointing the wrong way, you will run your samples into the buffer
• Add each of your samples to the wells
  • Use the entire sample
  • If possible, don't use adjacent wells in case of spill-over
  • Avoid using torn wells
• Place the lid on the gel box
  • Make sure the black cable is away from you and the red cable is near you
  • If you reverse the cables you will run your samples into the buffer
• Turn on the gel box
• Adjust voltage to ~100 V
• Wait until you can see that the 8X loading dye has moved 1–2 inches down the gel
  • If you can't see your loading dye, you've probably run your samples into the buffer

Gel slices

• Remove your gel from the box and take it to 758 WIDB
• Shine the UV lamp on your gel to determine where the bands are
• Cut out the bands using a razor blade
  • Carefully wipe the razor blade with a paper towel between each band
  • Cut out a DNA-free slice of the gel for your vector-only control
• Place slices in labeled microcentrifuge tubes
  

Note: These can be stored in the –20°C freezer

Ligation

• Put your slices (in microcentrifuge tubes) in the 65°C heating block for 3–5 minutes
• Prepare a Master Mix:
  • Remember to prepare an extra reaction for each vector-only control
  • 6.5 μL sterile ddH₂0 per rxn
  • 1.5 μL 10X T4 ligase buffer per rxn
    • Be sure to stir up the white chunks when thawing the buffer
  • 1.0 μL T4 ligase per rxn
• Bring the hot block with gel slices to your bench
• To empty 0.5-mL tube, add 3.0 μL of vector
• Add 3.0 μL of insert (or DNA-free slice if vector-only control)
• Add 9.0 μL Master Mix and mix (total reaction volume = 15 μL)
• Incubate at room temperature for at least 1 hour in the dark (i.e. in a drawer)

Note: At this point the samples may be stored in the –20°C freezer or you can remelt on the 65°C heating block (3–5 min) and proceed to transformation