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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 9 / 15

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Biol  420                                                                                                                                          Fall 2007

4.

Assemble the Gel Sandwich.

a.

Place the spacers on the back plate and then place the front plate on top of the rear plate by rotating the top plate onto the bottom plate.  Be sure that the plates are aligned at the bottom.

b.

Align the spacers with the outside edges of the plate.  Then place the left and right rail assemblies over the plate edges ( the top of the rail is identified by the groove where the upper buffer tank or casting plate is inserted).  Be sure that the rails fit tightly against the edges of the glass plates and spacers.  This will prevent leaks.

c.

Tighten the glass clamp knobs on each rail.  Be sure to tighten them only finger tight (just past the point of resistance.  Overtightening can break or distort the glass plate and may cause “smiles” on gel images due to uneven gel migration.

d.

In preparation for pouring the gel, place the prepared gel apparatus at a slight incline in the casting tray.

e.

Select the 0.2 mm sharkstooth comb and clean it with isopropanol if necessary.  Be sure that it fits between the two glass plates.  Let the comb sit nearby until you are ready to pour your gel.

5.

Prepare a 0.2mm, 66 cm gel by transferring 40 of the KBplus 3.7% Gel Matrix to a 200 ml beaker.  Allow the solution to come to room temperature by incubating on the bench top for 15 minutes.

a.

Add 230 ul of 10% APS (0.1 grams of APS dissolved in 1ml of ultrapure dH2O) and 23 ul of TEMED to the warmed Gel Matrix solution.

b.

Swirl the solution to mix well, collect all 40 ml into a syringe and carefully inject it into the gel cassette.

c.

Lay the gel flate and place flat side of sharkstooth comb into the top of the gel.  Be sure that the gel material it filled all the way to the top.

d.

Insert casting plate and tighten the top gel clamp knobs until finger tight.

6.

Let the gel polymerize for a minimum of 1.5 hours before use.

Loading the Samples and Running the gel.

1.

Once the gel has polymerized, loosen the upper knob on each rail and remove the casting plate.

2.

Add a small volume of water to the notched area on the front plate where the comb is inserted.  When the comb is removed water will be drawn into the wells which helps to maintain good well morphology.

3.

After removing the comb, a razor blade can be used to remove any excess gel from inside the back plate above the notched area where the comb was previously inserted.  Similarly remove the gel from the outside of the plates at the bottom and top of the gel sandwich.

4.

Use paper towels and dH2O to clean the back and front plates.  The region where the sequencer will scan must be carefully cleaned.

5.

Then reinsert the sharkstooth comb just until the teeth touch the gel.  Hold the gel upright against a good light source so that the bottom of the well is clearly visible.

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