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

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&mdash;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
Note: These can be stored in the –20°C freezer
 * 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

Ligation
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
 * 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)