On the day before this lab, a large quantity of the plasmid containing the plasmid rescue fragment for the common mutant will be digested. The digested DNA will be size fractionated on a gel before class starts. During class each group will cut out the bands of the plasmid resuce DNA from two lanes. This DNA will be removed from the agarose and purified in preparation for labeling. We will use a commercially available Gene Clean™ Kit (Obiogene). Follow the instructions below.

1. Determine the approximate volume of the gel slice by weight (1 g equals approximetely 1 ml). Every mg of weight is equivalent to 1 µl of solution. Add 3 volumes of NaI to the tube. For example, if your gel weighs 300 mg, add 3 x 300 µl of NaI.
2. Place the tube in a 45 - 55 ^oC water bath for 5 minutes. Check to be sure that all of the gel has melted.
3. While the gel is melting, re-suspend the glassmilk by vortexing.
4. Add 5 µl glassmilk to the gel + NaI solution.
5. Mix and incubate at room temperature for 5 minutes to allow binding of the DNA to the silica gel. Invert the tube every couple of minutes to keep the silica suspended.
6. Pellet the silica matrix with the bound DNA by spinning the microcentrifuge tube for 5-10 seconds.
7. Remove the NaI. Re-suspend the pellet in New Wash. Pellet and remove the wash solution. Do this 3X.
8. After removing the New Wash the third time pellet the glassmilk again and remove any residual liquid.
9. Add 5-10 µl of sterile distilled water or Elution Solution from the kit. Pellet the glassmilk. This time remove the solution and move to a new tube. Be careful not to transfer any of the glassmilk.
10. Run 1 µl of the purified DNA on a test gel.