Griffitts:In-gel ligation

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Contents

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

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

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