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Buffer gradient SEQUENCING GELS

1. Wash new plates thoroughly using detergent and dry. Wipe each plate with 100% ethanol. Wipe the plate with ears only using Rain-X to siliconize.

2. Clamp plates together, remembering to place a little vacuum grease between side and bottom spacers, and place spatula to spread plates slightly at the top to facilitate pouring the gel.

3. To pour your gel you need

100 ml measuring cylinder


25% APS

2 bottles for pouring


2.5 x TBE sequencing buffer

0.5 x TBE sequencing buffer

35ml pipette and bulb

For a 60cm buffer gradient gel:

a) To one of the bottles add 74 mls of 0.5 X TBE gel buffer.

b) Using the same measuring cylinder, add 21 mls of 2.5 X TBE gel buffer to the second bottle.

To "a" add 130ul of 25% APS and 85ul to "b". Swirl gently.

To "a" add 40ul of TEMED and 25 ul to "b". Swirl gently.

4. Suck up 10ml of 0.5 x, then all of the 2.5 x in the pipette. Be careful not to mix the 0.5 x and 2.5 x in the pipette (this is to form a discontinous gradient). Lift plates and slowly pipette this in. As it finishes lower plate so that a column of gel solution extends up the plate. Pour in remainder of 0.5 x solution, maintaining a continuous stream. An uninterupted flow is important to avoid bubbles and prevent the gradient being displaced when the bulk of the 0.5 x flows in.

5. Push in shark tooth comb about 4mm.

6. When the gel is set remove clamps, bottom spacer and comb. Wash comb area and push teeth of comb into gel. Mark any areas that have bubbles so you don't load any samples in this region. Fill tanks with 0.5 x TBE.

7. Remove air bubbles where the bottom spacer was, using a syringe and needle full of 0.5 x TBE running buffer. Flush comb out before loading the gel.

8. Pre-run gel at 55W (constant power) for 15 minutes.

2.5 x TBE gel mix for sequencing

230 gms urea

62.5 mls 20 x TBE

75 mls 40% acrylamide (19:1 and without urea)

25 gms sucrose

25 mg Bromophenol blue

DDW to 500 mls

0.5 x TBE gel mix for sequencing

460 gms urea

25 mls 20 x TBE

140 mls 40% acrylamide no urea

DDW to 1 litre.

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This page is maintained by David Bowtell ( using HTML Author. Last modified on 10/24/95.