exercise 4:
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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 March 10. - PowerPoint PPT PresentationTRANSCRIPT
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Exercise 4:
DNA
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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.
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Goals
• Purify chromosomal DNA from E. coli.• Map the sites for the restriction
endonucleases BamHI and HindIII on plasmid pBR322 DNA.
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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.
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Genome Size Varies Widely
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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.
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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.
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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
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Map of pBR322
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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.
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Naming of Restriction Enzymes
• Named for the organism of origin.
– BamHI was isolated from Bacillus amyloliquefaciens
– HindIII was isolated from Haemophilus influenzae
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Restriction Enzymes may require specific buffers:
• Buffers adjusted to optimal:– pH– Ionic strength– Mg concentration
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Joining Restriction Fragments
Restriction fragments can be joined by the enzyme DNA ligase
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• Used to tell which regions of a cloned gene could be sub-cloned for over-expression of a particular protein.
Restriction Maps
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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
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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.
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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:
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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: