Sequencing Gel

Each sequencing gel contains:

Urea                            21g                              2l of 10*Sanger TBE contains

Sequagel XR               6 ml                             TRIS base       324 g

10*Sanger TBE           5 ml                             Boric acid        85 g

APS                             0.05g                           EDTA             19 g

TEMED                      20 ml                            Make up to 2L with water

Make up to 50 ml with water

1.    Weigh out and mix the following in a conical flask: 21g urea, 0.05g APS, 6 ml Long Ranger, and 5ml 10xTBE.

2.    make up to 50 ml with water (use approx. 25 mls of water).

3.    Melt the urea by putting the flask under running hot water

4.    Allow the solution to cool for around 10 minutes and put in 20 ul TEMED.

5.    The gel is now ready for pouring.

Pouring of gel

1.    Select a long and a short glass plate. Clean thoroughly with methanol and then acetone.

2.    Put the white spacers onto the long plate and then cover with the short plate. Put clips around both sides of the plates.

3.    Pour the gel solution slowing into the exposed end of the long plate (the TEMED must just be added before). Tap the plate gently to remove any air bubbles which may form.

4.    Insert the comb (straight end) onto the gel apparatus.

5.    Place clips around the top of the gel to fix the comb

6.    Allow the gel to set for at least 30 minutes.

Assembley of gel apparatus

1.    After the gel has set, remove the clips and place the plates (short plate towards the back) onto an electrophoretic tank. Attach the plates to the tank with clips and spacers.

2.    Pour 1*Sanger TBE onto the top reservoir (make sure that the drain tap is closed) and observe for any leakage. In the event of leakage, try to seal the leak with vasoline.

3.    Take the comb out of the gel, and clear the area of acrylamide.

4.    Using a syringe, blow buffer onto the top part of the gel to clear clumps of acrylamide

5.    Re-insert the comb with the toothed edges down

Loading of gel

1.    Place the microtitre plate containing the samples onto a heating block at 100^oC for 5 minutes

2.    Label long plate with the name of the samples

3.    Fill the top and bottom reservoirs of the gel tank with 1*Sanger TBE

4.    Load the 4 ml of each sample onto the appropriate well. Observe for any leakage onto adjacent wells.

5.    Plug into the power supply and run the gel at 75W until the samples has gone into the gel.

6.    After the samples have gone into the wells, take the comb out and run the gel at 75W for about one hour until the dark blue dye reaches the bottom.

Removal and drying of gel

1.    At the end of electrophoresis turn off the power and open the gel tank drain.

2.    Take the plates out and lay down flat (long plate at bottom)

3.    Remove the spacers

4.    Carefully prise the plates apart, note which plate the gel stays on.

5.    Absorb the gel onto a large piece of filter paper and cut the paper to size.

6.    Wrap the filter the gel on filter paper with Saran wrap.

7.    Heat at 80^oC under vacuum and for 1-2 hours

Autoradiography

1.    Affix dried gel to a film cassette by tape

2.    In the darkroom, insert an X-ray film into the cassette, with the notch on the top left corner

3.    Leave overnight and develop the film.

4.     Examine the developed film through an X-ray box, making sure that the side is correct.