Exercise 4:

DNA

Announcements

• Post Lab 4 and Pre Lab 5 are due by your next lab period.

• LNA: This weeks lab and next weeks go together. Be sure to write your procedures, and any changes made. It will not be due until the week of October 12.

• *You must be present for both Exercises 4 and 5 in order to turn in the Lab Notebook Assignment for credit. If you were absent for either week you will earn a zero on this assignment.

Goals

• Purify chromosomal DNA from E. coli.• Map the sites for the restriction

endonucleases BamHI and HindIII on plasmid pBR322 DNA.

The E. coli Chromosome

• Single, large, circular DNA molecule.• About 1 mm long• Genome ~ 4 x 106 bp (base pairs)• Consists of ~ 50% A-T bp and ~ 50% G-C bp• Since the average gene is ~ 1000 bp, E. coli

encodes ~ 4000 proteins.

Genome Size Varies Widely

Purification of Chromosomal DNA

Step:1. Disrupt the cell membrane, lysing the

cells.2. DNA molecules become susceptible to

shear force which break the DNA into linear fragments. (20-30 kb)

3. Precipitate the DNA.

Isolating Chromosomal DNA from E. coli

1. Lyse cells with sodium dodecyl sulfate.2. Degrade proteins with Proteinase K.3. Extract DNA with chloroform.4. Precipitate DNA with 95% EtOH.5. Collect DNA by winding fibers around a glass rod.6. Dissolve the DNA in Tris-HCl buffer + EDTA.7. Analyze by gel electrophoresis.

Plasmids

• Self-replicating, extrachromosomal DNA• Most are double stranded• Circular DNA• Supercoiled• Size: 2 kb - several hundred kb• Vary in the number of copies/cell

Map of pBR322

Restriction Enzymes

• Recognize and cut specific sequences in double-stranded DNA.

• The longer the recognition sequence the lower the probability of finding that specific sequence.

• Since there are 4 bases, the probability of finding a specific sequence is 1/4n

Where n is the number of nucleotides.

Naming of Restriction Enzymes

• Named for the organism of origin.

– BamHI was isolated from Bacillus amyloliquefaciens

– HindIII was isolated from Haemophilus influenzae

Restriction Enzymes may require specific buffers:

• Buffers adjusted to optimal:– pH– Ionic strength– Mg concentration

Joining Restriction Fragments

Restriction fragments can be joined by the enzyme DNA ligase

• Used to tell which regions of a cloned gene could be sub-cloned for over-expression of a particular protein.

Restriction Maps

Making a Restriction Map(double digests)

• Take 3 aliquots of purified DNA and treat with two different enzymes.

1. Treat aliquot #1 with enzyme #12. Treat aliquot #2 with enzyme #23. Treat aliquot #3 with enzymes #1 and #2

• Compare the resulting sets of fragments by gel electrophoresis

Nucleases

• Purified DNA is very sensitive to nucleases, and can degrade rapidly if a nuclease is present.

• Where gloves to prevent your own nucleases from degrading your sample.

Isolating Chromosomal DNA from E. coli

1. Lyse cells with sodium dodecyl sulfate.2. Degrade proteins with Proteinase K.3. Extract DNA with chloroform.4. Precipitate DNA with 95% EtOH.5. Collect DNA by winding fibers around a glass rod.6. Dissolve the DNA in Tris-HCl buffer + EDTA.7. Analyze by gel electrophoresis. (Week 5)

Part I:

Restriction Analysis of Plasmid DNA

1. Set up 4 digests (EcoRV, PstI, EcoRV+PstI, uncut).2. Cover your digests, flick the bottoms to mix, and

centrifuge.3. Incubate at 37C for 1 hour.4. Stop reactions by adding 5x Blue loading solution.5. Analyze by gel electrophoresis. (Week 5)

Part II: