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Exercise 4:

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.

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Exercise 4:

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  1. Exercise 4: DNA

  2. 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. • *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.

  3. Goals • Purify chromosomal DNA from E. coli. • Map the sites for the restriction endonucleases BamHI and HindIII on plasmid pBR322 DNA.

  4. 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.

  5. Genome Size Varies Widely

  6. Purification of Chromosomal DNA Step: • Disrupt the cell membrane, lysing the cells. • DNA molecules become susceptible to shear force which break the DNA into linear fragments. (20-30 kb) • Precipitate the DNA.

  7. Isolating Chromosomal DNA from E. coli • Lyse cells with sodium dodecyl sulfate. • Degrade proteins with Proteinase K. • Extract DNA with chloroform. • Precipitate DNA with 95% EtOH. • Collect DNA by winding fibers around a glass rod. • Dissolve the DNA in Tris-HCl buffer + EDTA. • Analyze by gel electrophoresis.

  8. 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

  9. Map of pBR322

  10. 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.

  11. Naming of Restriction Enzymes • Named for the organism of origin. • BamHIwas isolated from Bacillus amyloliquefaciens • HindIII was isolated from Haemophilus influenzae

  12. Restriction Enzymes may require specific buffers: • Buffers adjusted to optimal: • pH • Ionic strength • Mg concentration

  13. Joining Restriction Fragments Restriction fragments can be joined by the enzyme DNA ligase

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

  15. Making a Restriction Map(double digests) • Take 3 aliquots of purified DNA and treat with two different enzymes. • Treat aliquot #1 with enzyme #1 • Treat aliquot #2 with enzyme #2 • Treat aliquot #3 with enzymes #1 and #2 • Compare the resulting sets of fragments by gel electrophoresis

  16. 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.

  17. Isolating Chromosomal DNA from E. coli • Lyse cells with sodium dodecyl sulfate. • Degrade proteins with Proteinase K. • Extract DNA with chloroform. • Precipitate DNA with 95% EtOH. • Collect DNA by winding fibers around a glass rod. • Dissolve the DNA in Tris-HCl buffer + EDTA. • Analyze by gel electrophoresis. (Week 5) Part I:

  18. Restriction Analysis of Plasmid DNA • Set up 4 digests (EcoRV, PstI, EcoRV+PstI, uncut). • Cover your digests, flick the bottoms to mix, and centrifuge. • Incubate at 37C for 1 hour. • Stop reactions by adding 5x Blue loading solution. • Analyze by gel electrophoresis. (Week 5) Part II:

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