1. Genomic Complexity

2. How many genes in the chromosome?
DNA or RNA?
Double Stranded or Single Stranded?
Shape?
Number of copies?
Size in base pairs?
Assume 400 amino acids per protein, then how many bases per gene for a typical protein?
How many genes possible?
Do genes overlap?
Are both strands transcribed?

3. Consider the Ribosome

4. And its operon

5. Studying Macromolecules –Extracting DNA
Cell Pellet
Random fragments
~20-40 kbp

6. Extraction of Chromosomal DNA
DNeasy Tissue Kit
Chromosomal DNA, ~ kbp size fragments
Kim Foster
23 kbp
Chromosomal DNA
RNA
Chromosomal DNA
RNA

7. Hybridization

8. Quantitative Hybridization

9. Liquid Scintillation Counters
Spike
Photomultiplier Tubes
Electronic Noise
Explain how the PMT tubes reject noise and have to have identical pulses to be recorded. Explain how sample volume and efficiency are related. Explain that the cocktail emits light and that if the particle is near the edge it is small and ends up lower on the scale. Explain that if two flashes occur at exectly the same time, it appears as one and ends up high on the scale. This increases as the activity in the sample increases. Explain how this relates to each of the spectrums and how the windows are set up. Just because there is activity in the tritium channel does not necessarily mean that there is tritium in the sample. It may be due to P-32. 3H
14C
32P
log Scale

10. Experiment 1 – Total 14C-Uracil
10 µg RNA
5 µg RNA sticks
10 µg DNA
5 µg RNA washes off

11. Experiment 2 – rRNA 14C-Uracil
10 µg rRNA
35 ng rRNA sticks
10 µg DNA
>99% rRNA washes off

12. Experiment 3 – tRNA 14C-Uracil
10 µg tRNA
5 ng tRNA sticks
10 µg DNA
>99% tRNA does not stick