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 7.
• *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
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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.
2.
3.
4.
5.
6.
7.
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.
Plasmids
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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
Compatible sticky ends -- base-pairing can occur:
BamHI
GATCC
G
G
CCTAG
BamHI
Incompatible sticky ends -- base-pairing cannot occur:
BamHI
AATTC
G
G
CCTAG
EcoRI
Blunt ends can always be joined together since no base-pairing
is involved:
EcoRV
GATATC
CTATAG
original site
Note
GAT GGG
CTA CCC
SmaI
CCCGGG
GGGCCC
original site
The original restriction sites are not reformed in this
recombined site
Restriction fragments can be joined by the enzyme DNA ligase
Restriction Maps
• Used to tell which regions of a cloned
gene could be sub-cloned for overexpression of a particular protein.
Making a Restriction Map
(double digests)
•
Take 3 aliquots of purified DNA and treat
with two different enzymes.
1. Treat aliquot #1 with enzyme #1 (digest)
2. Treat aliquot #2 with enzyme #2 (digest)
3. Treat aliquot #3 with enzymes #1 and #2
(double digest)
•
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
Part I:
1.
2.
3.
4.
5.
6.
7.
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)
Restriction Analysis of Plasmid
DNA
Part II:
1.
2.
3.
4.
5.
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)