1. Lab 20 Goals and Objectives: Exercise 58: Bacterial Counts on Foods Count colonies: calculate CFU/gram (meat, spinach) or CFU/ml (milk) *Remember, plates of 30-300 only! Share data, get class averages Exercise 59: Bacteriological Examination of Water Determine the MPN (Table 59.1) coliforms /100ml Streak one positive tube for isolation on EMB agar Exercise 60: The Membrane Filter Method Check filters for coliforms: (examples of Escherichia and Enterobacter on EMB provided to demonstrate appearance of coliforms) Count coliforms and compare to MPN from Ex 59 Biotechnology Practice Exercise Each pair get: Two Pipettors, Box of tips, Tube of colored water, Two squares of parafilm: Practice pipetting 5µl water: make a 3 X 3 5µl drop pattern on the parafilm and check that all drops look the same size EDVOKIT#300: Blue/White Cloning of a DNA Fragment Work in five groups total (double up with another pair?) Set up the ligation (tube 1) and ligation control (tube 2) reactions according to directions on pg 11. When done, put in rack to incubate.

2. To “clone a gene”: means to copy the gene from its original natural genomic source and to put it into a plasmid/vector for expression (protein product) or other experimental use.

3. EDVO page 4

4. EDVO page 5 Restriction enzymes cut DNA at certain base sequences leaving “sticky ends” that “want” to ligate with other complementary sticky ends.

5. Vector + gene we want to clone + ligase ~incubate~ Two possible products: -gene ligated into vector -vector religated without gene 

6. Ligation Transformation EDVO page 6 Experiment Overview

7. Biotechnology Tools Micropipettor Practice Good consistent pipetting! Need more practice! - touch tip to surface when dispensing - “blow out” sample and pull away before letting go of plunger

8. EDVO page 11 Set up ligation