Gel Electrophersis

2. Steps for Southern Blotting

* Digest the DNA with an appropriate restriction enzyme.

* Run the digest on an agarose gel.

* Denature the DNA, which would separate double-stranded DNA into single-stranded DNA.

* Transfer the denatured DNA to the membrane.

* Probe the membrane.

* Visualize your radioactively labeled target sequence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph.

3. a) She placed the nucleotide as she did because the EcoR I cuts in one place which is why there are two bands. One fragment is slightly smaller than the other.

b) She was able to add this sequence because The Hind III by itself only cut once. The EcoR I also cut once. In the double digest, the Hind III cuts the smaller of the EcoR I fragments.

4. The odds that the sequence will occur in random order is 1 out of 4096. Since there are four bases possible and they must appear in a six base sequence the answer is 1 out of 4.

5. By digesting the DNA with the above enzyme there would be about then divide 3 billion by 4,096

7. Advantages bacteria gain by having these restriction enzymes is that restriction enzymes serve as a means of defense for a bacterial cell. Restriction enzymes can prevent DNAfrom other organisms, such as bacterial viruses or other bacterial species, from entering the cell and taking over vital cell processes.

9. If you placed the gel with the wells next to the red electrode instead of the black one is that the DNA would still be attracted to the positive electrode. The DNA samples would run quickly toward the top of the gel and out into the buffer.

10. Gel only slightly higher percentage of Agarose would probably not affect the results significantly. However, if the percentage were greatly increased, the larger fragments may not be able to move through the decreased pore size making it difficult, resulting in less effective electrophoretic separation.

11. Agarose gel - the substrate used to separate molecules. Contains microscopic pores that allow

smaller molecules to move through faster.

TBE buffer - a liquid buffer that protects the DNA molecules and allows electricity to move

through