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