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Molecular Biology
Working with DNA
Topics
Genomic vs. Vector DNA
 Purifying plasmid DNA
 Restriction enzymes
 Restriction maps

DNA

Genomic




Prokaryote vs. eukaryote
Circular or linear
One or more chromosomes
Extra-genomic


Vectors
Plasmids
Vectors Vs Plasmids

Vector:



DNA vehicle that allows the cloning, maintenance
and amplification of a DNA sequence
 Plasmids
 Virus
 Chromosomes
All plasmids are vectors
Not all vectors are plasmids
Plasmids

Small circular DNA molecules maintained and
amplified in eukaryotic or prokaryotic cells


Amplification in bacteria
Used as vector for cloning or expression of
DNA of interest
Characteristics of plasmid vectors
Restriction sites for
cloning
 Origin of replication
(Ori)
 Selection marker


Genes conferring
resistance to antibiotics
DNA Isolation

Goals


Isolation of DNA of interest
 Chromosomal or plasmid?
Eliminate other components
 Chromosomal or plasmid DNA?
 Proteins
 RNA
 Chemicals
Salts, detergents, etc.
DNA isolation

Cell lysis


Cell wall and membrane
 Enzymatic
 Chemical
 Mechanical
Isolation of DNA of interest



(cont’d)
Differential sedimentation
Chromatography
Removing other components



Enzymatic
Differential sedimentation
Chromatography
Plasmid DNA isolation by alkaline
lysis (E.coli )
Solutions Used

Sol. I – Resuspension buffer



Tris HCl – Buffer that protects nucleic acids
EDTA - Chelates Mg++, prevents nucleases from
working
Sol. II – Lysis solution


NaOH - ^pH lyses cells, denatures DNA
SDS – Dissolves membranes, denatures and binds
proteins
Solutions Used

Sol. III- Potassium acetate




Renaturation of DNA
Precipitates SDS
Precipitates genomic DNA and proteins
Isopropanol / Ethanol



(Cont’d)
Precipitates nucleic acids (plasmid and ?)
Salts remain soluble
TE-RNase - Tris & EDTA again; RNase??
Quantification of DNA

Determining Conc. of DNA


Determining Amount of DNA



A260 of 1.0 = 50µg/mL or 50ng/µL
1mL of a solution with an A260 of 1.0 contains 50µg DNA
1µL of a solution with an A260 of 1.0 contains 50ng DNA
Do not forget to account for the DILUTION FACTOR
Restriction enzymes

Endonuclease




Cleaves internal phosphodiester linkages
Recognize specific double stranded DNA
sequences
Different endonucleases recognize different
sequences
Recognize palindrome sequences
Palindromes

The same sequence is read in the 5’ » 3’
direction on both strands
5’-G G A T C C-3’
3’-C C T A G G-5’

The same phosphodiester linkages are
cleaved on both strands!
5’-G G A T C C-3’
3’-C C T A G G-5’
Different ends are generated
5’-G G A T C C-3’
Blunt ends
3’-C C T A G G-5’
Different ends are generated
5’-G G A T C C-3’
5’ overhangs
3’-C C T A G G-5’
Different ends are generated
5’-G G A T C C-3’
3’ overhangs
3’-C C T A G G-5’
Compatibility of ends
Blunt ends
P
OH
HO
P
OP
PO
Compatible
Compatibility of ends
Overhangs
P
OH
P
HO
OP
P
HO
Incompatible
Compatibility of ends
Overhangs
GATC-P
HO
P-CTAG
OH
Annealing
GATC-P O
O P-CTAG
Compatible
Compatibility of ends
Overhangs
GATC-P
HO
P-TCCA
OH
Annealing
GATC-P
OH
HO
P-TCCA
Incompatible
Restriction Maps
Restriction maps

Determining the positions of restriction
enzyme sites



Linear DNA maps
Circular DNA maps (plasmids)
Maps of inserts within vectors
Approach
Determine whether the DNA has digested
2. Is the digestion complete or partial?
3. How many cuts?
4. Determine the relative positions
1.
1. Is the DNA digested?
1
2
3
4

Compare to the
undigested control

Which samples were
not digested?


1 and 4
Which samples were
digested?

2 and 3
2. Is the digestion complete?

Complete digestion


All the DNA molecules are cleaved at all the possible
sites
Partial digestion


A fraction of the molecules are not digested
 Partial undigested
A fraction of the molecules were digested, but not at
all the possible sites
 Partial digestion
Complete digestion
Digestion
Partial digestion: Partial undigested
Non digested
Digestion
Partial digestion
Digestion
partial
partial
Is the digestion complete or partial?
1
2
3
4



Compare to control
Verify the intensity
of the bands
Verify the sizes
3. How many cuts?

Number of sites


Circular DNA = number of bands
Linear DNA = Number of bands – 1
4. Determine the relative positions
 The fragment sizes represent the distances
between the sites
Linear DNA maps
Enzyme
Fragments (Kb)
HindIII
3 and 4
SalI
2 and 5
HindIII + SalI
2 and 3
7.0
3.0
HindIII
4.0
3.0
2.0
2.0
HindIII + SalI
Circular DNA maps (plasmids)
Enzyme
Fragments (Kb)
BamHI
2, 3 and 5
HindIII
1 and 9
BamHI + HindIII
1, 1.5, 2, 2.5 and 3
7.0
10.0
1.5
10.0
Insertion maps
Insertion site
MCS
MCS
Recombinant plasmid
Vector
Approach
Determine the total size
2. Determine size of the insert
1.

Total size – size of vector
Determine the insertion site within the MCS
4. Determine which enzymes cut wihin the
insert
5. Relative mapping in relation to the sites at
known positions
3.
Insertion maps
1.
Total size
•
2.
Insert size
•
Enzyme
Fragments
BamHI
7.7Kb
EcoRI
1.0, 3.0, 3.7Kb
PstI
2.0 and 5.7
XbaI
2.7 and 5.0
3.
7.7Kb
7.7 – 2.7 = 5.0Kb
Insertion site
•
Generates 2 fragments
of which one is the size
of the vector
•
XbaI
Insertion maps
Sites to map
Enzyme
Fragments
Total cuts
Sites in
vector
Sites in
insert
BamHI
7.7Kb
1
1
0
EcoRI
1.0, 3.0, 3.7Kb
3
1
2
PstI
2.0 and 5.7
2
1
1
XbaI
2.7 and 5.0
2
Insertion
site
0
Map of PstI : 2 and 5.7Kb
5.7 Kb
2.0 Kb
5.0
Map of EcoRI: 1, 3 and 3.7Kb
P
3.7
1.0
3.0
1.0
1.0
3.0
Micropipetting-Measuring small volumes

Allows to measure microliters (µL)


1 000 X less than 1 milliliter
2-20 µL
Max. 0.02 mL
50-200 µL
0.2mL
100-1000 µL
1mL
Setting the volume- P20
Tens (0, 1=10 or 2=20)
Units (0-9)
Decimal (1-9 = 0.1-0.9)
Setting the volume- P200
Hundreds (0, 1=100 or 2=200)
Tens (0, 1-9=10-90)
Units (1-9)
Setting the volume- P1000
Thousands (0, 1=1000)
Hundreds (0, 1-9=100-900)
Tens (0, 1-9=10 - 90)
Using the micropipettor
Step 1
Insert tip
Step 2
Press plunger
up to first stop
Step 3
Insert tip in solution to be
drawn
Step 4
Draw up sample by
slowly releasing plunger
Step 5
Withdraw pipettor
Dispensing
Start dispensing
1st stop =Dispense 2nd stop = Expel
Guidelines for optimal reproducibility
Use pipettor whose volume is closest to the one
desired
 Consistent SPEED and SMOOTHNESS to press and
release the PLUNGER
 Consistent IMMERSION DEPTH


3-4mm below surface
AVOID air bubbles
 NEVER go beyond the limits of the pipettor
