Announcements
• Lab Exam 12/10 – 12/12 during discussion
• ~20 multiple choice questions
• Will require a calculator
• Extra Room for Wed. Section: KCB 107
• All Chapter 6 Labs Due by 12 noon on Wed. 12/12
Discussion Dates
Lab Dates
Chapter 6ab
11/26 – 11/28
11/28 – 12/3
Chapter 6c
12/3 – 12/5
12/5 – 12/10
Lab Exam
12/10 – 12/12
Lab Due Dates
All Sections
Noon, 12/12
Boxes outside SCI 162
Chapter 6: Week 2 – Restriction
Digest of Plasmid DNA
Purpose:
1) Learn about restriction enzymes and
plasmid maps
2) Perform restriction enzyme digest to
identify your plasmids
Restriction Enzymes
●
Restriction Endonucleases
●
●
●
●
Recognize and cleave DNA to make smaller fragments
DNA fragments can be cloned into new molecule using DNA
ligases
Often protected from digestion in the cell by DNA
methylation
3 Types of Restriction Enzymes:
●
●
●
Type I: Cleave DNA at random sites, > 1000 bp from
restriction sequence, requires ATP
Type II: Cleave DNA within recognition sequence, does not
require ATP
Type III: Cleave DNA about 25 bp from recognition sequence,
requires ATP
Type II: Restriction Enzymes
●
●
●
Only cut DNA at specific
recognition sequences
Recognition sequences
typically 4-6 bp long
Often palindromic – Dyad
Symmetry
EcoRI: Yields products with 5’
overhangs that can base pair
with each other
5’ –GAATTC– 3’
3’ –CTTAAG– 5’
Phosphodiester
Bond Cleavage
EcoRV in complex with
DNA (1RVC)
-2O PO-AATTC– 3’
5’ –G-OH
3
3’ –CTTAA-OPO32HO-G– 5’
Type II: Restriction Enzymes
●
Restriction Enzymes can give:
●
●
●
5’ Overhangs: EcoRI
5’ –GAATTC– 3’
3’ –CTTAAG– 5’
3’ Overhangs: PstI
5’ –CTGCAG– 3’
3’ –GACGTC– 5’
Blunt Ends: PvuII
5’ –CAGCTG– 3’
3’ –GTCGAC– 5’
Overhangs are
often called
“Sticky Ends”
Type II: Restriction Enzymes
●
Restriction Enzymes can give:
●
●
●
5’ Overhangs: EcoRI
5’ –G-OH -2O3PO-AATTC– 3’
3’ –CTTAA-OPO32- HO-G– 5’
3’ Overhangs: PstI
5’ –CTGCA-OH -2O3PO-G– 3’
3’ –G-OPO32- HO-ACGTC– 5’
Blunt Ends: PvuII
5’ –CAG-OH -2OPO3-CTG– 3’
3’ –GTC-OPO32- HO-GAC– 5’
What are the products of a
restriction enzyme digest?
●
Digest DNA with RE
1.0
Run gel
0.9
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Observe fragmentation of DNA
0.8
●
●
Plot migration distance (mm) of
standards vs. Log fragment size
Use graph to find size of
fragments, see p. 192
Fragments: 17.5 mm, 22.0 mm
5.42 kb, 3.47 kb
●
Find total size of plasmids by
adding up the fragments
Log Fragment Size (kbp)
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0.7
0.6
0.5
0.4
0.3
0.2
y = -0.0432x + 1.4906
R² = 0.997
0.1
0.0
0
10
20
30
Migration Distance (mm)
40
Plasmid Maps
●
●
Used to determine location of
restriction enzyme sites on
plasmid
Perform restriction enzyme
digest, run gel, measure
fragments:
●
●
●
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EcoRI
HindIII
EcoRI +
HindIII
Marker
8kb
7kb
6kb
5kb
EcoRI: 3 kb, 5 kb
HindIII: 2 kb, 6 kb
EcoRI + HindIII:
2 kb, 1 kb, 5 kb
Total Size of Plasmid: 8 kb
4kb
3kb
2kb
1kb
Plasmid Maps
●
●
Used to determine location of
restriction enzyme sites on plasmid
HindIII, EcoRI
0 kb (8 kb)
Perform restriction enzyme digest,
run gel, measure fragments:
●
EcoRI: 3 kb, 5 kb
●
HindIII: 2 kb, 6 kb
●
●
EcoRI + HindIII:
2kb, 1 kb, 5 kb
2
HindIII
2 kb
Plasmid X
(8 kb)
Total Size of Plasmid: 8 kb
EcoRI
3 kb
Plasmid Maps: Pop Quiz
Digestion
Fragment Size (bp)
BamHI
2800
EcoRI
2800
HindIII
2800
BamHI + HindIII
1800, 1000
HindIII + EcoRI
1600, 1200
EcoRI + BamHI
2600, 200
Construct the restriction
enzyme map for this plasmid
HindIII
0 bp
Plasmid Maps: Pop Quiz
Digestion
Fragment Size (bp)
BamHI
2800
EcoRI
2800
HindIII
2800
BamHI + HindIII
1800, 1000
HindIII + EcoRI
1600, 1200
EcoRI + BamHI
2600, 200
HindIII
0 bp
Construct the restriction
enzyme map for this plasmid
No single cuts in plasmid
Therefore, use 1st double cut
2800-1800 = 1000 bp
2800-1000 = 1800 bp
BamHI
1000 bp
Plasmid Maps: Pop Quiz
Digestion
Fragment Size (bp)
BamHI
2800
EcoRI
2800
HindIII
2800
BamHI + HindIII
1800, 1000
HindIII + EcoRI
1600, 1200
EcoRI + BamHI
2600, 200
HindIII
0 bp
Construct the restriction
enzyme map for this plasmid
Use EcoRI + BamHI:
2800-2600 = 200 bp
2800-200 = 2600 bp
Should be directly next to
BamHI site
BamHI
EcoRI 1000 bp
1200 bp
Plasmid Maps: Pop Quiz
Digestion
Fragment Size (bp)
BamHI
2800
EcoRI
2800
HindIII
2800
BamHI + HindIII
1800, 1000
HindIII + EcoRI
1600, 1200
EcoRI + BamHI
2600, 200
HindIII
0 bp
Construct the restriction
enzyme map for this plasmid
Check math with last double digest:
2800-1600 = 1200 bp
Plasmid Map complete!
2800-1200 = 1600 bp
BamHI
EcoRI 1000 bp
1200 bp
Identifying Our Plasmids
●
Using your restriction digest gel,
identify fragments from by size
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PvuII
●
AhdI
●
SP6
HindIII
pGEM3
REL
AmpR
AhdI
PvuII
BamHI
ORI
PvuII
PvuII + AhdI
SP6
BamHI
PvuII
●
●
How many fragments should you
have in each lane?
Identify which plasmid is which
by differences in size of two PvuII
sites
pGEM4
REL
AmpR
AhdI
ORI
PvuII
HindIII
T7
Procedure: Chapter 6 – Week 2
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Restriction Enzyme Digest
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Agarose Gel Electrophoresis
If you are taking Biochemistry 2, make sure to
label and save your plasmids for next semester!
Procedure: Chapter 6 – Week 2
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Restriction Enzyme Digest
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Prepare samples in 0.5 ml centrifuge tubes:
Single Digestions (x4)
1 µl 10 X Buffer 4 (NEBL)
Double Digestions (x2)
1 µl 10 X Buffer 4 (NEBL)
2 µl plasmid DNA (~0.5 µg)
6.5 µl Water (change with DNA)
0.5 µl PvuII or AhdI
2 µl plasmid DNA (~0.5 µg)
6 µl Water (change with DNA)
0.5 µl of PvuII and AhdI
10 µl Total Volume
10 µl Total Volume
●
Estimate DNA mass from agarose gel from week 1
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Vortex, Digest at 37°C for 1 hr
Procedure: Chapter 6 – Week 2
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Agarose Gel Electrophoresis
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Prepare Gel:
–
●
While digest is running, pour 1% agarose gel (1 gel/ group)
Sample Preparation:
Single Digestions X 4
2 µl 6X Sample Buffer
10 µl of Single Digest
12 µl Total Volume
●
Double Digestions X 2
2 µl 6X Sample Buffer
10 µl of Double Digest
12 µl Total Volume
Load Gel:
–
6 samples and 1 standard / gel
–
Standard: Linear DNA Minnesota Molecular (Table II, p. 184)
Procedure: Chapter 6 – Week 2
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Agarose Gel Electrophoresis
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Run Gel:
–
What is charge on DNA? Which direction will it run?
–
Run gel at 100-125 V until dyes separate and are near bottom
of gel
–
Record volts, amps, running time, etc. in your lab notebook
Staining and De-staining of Gel:
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Stain in ethidium bromide, 10 – 15 min
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De-stain in water, 1 min
Image Gel:
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Take picture of agarose gel on gel dock