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 Lab Due Dates
Chapter 6ab 11/26 – 11/28 11/28 – 12/3
Chapter 6c 12/3 – 12/5 12/5 – 12/10 All Sections
Noon, 12/12
Boxes outside SCI 162
Lab Exam 12/10 – 12/12
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
EcoRV in complex with
with each other
DNA (1RVC)

5’ –GAATTC– 3’ Phosphodiester 5’ –G-OH -2O PO-AATTC– 3’

3
3’ –CTTAAG– 5’ Bond Cleavage 3’ –CTTAA-OPO32- HO-G– 5’
 Type II: Restriction Enzymes
● Restriction Enzymes can give:

5’ –GAATTC– 3’
● 5’ Overhangs: EcoRI
3’ –CTTAAG– 5’
Overhangs are
often called
“Sticky Ends”
5’ –CTGCAG– 3’
● 3’ Overhangs: PstI 3’ –GACGTC– 5’

5’ –CAGCTG– 3’
● Blunt Ends: PvuII 3’ –GTCGAC– 5’
 Type II: Restriction Enzymes
● Restriction Enzymes can give:

5’ –G-OH -2O3PO-AATTC– 3’
● 5’ Overhangs: EcoRI
3’ –CTTAA-OPO32- HO-G– 5’

5’ –CTGCA-OH -2O3PO-G– 3’
● 3’ Overhangs: PstI 3’ –G-OPO32- HO-ACGTC– 5’

5’ –CAG-OH -2OPO3-CTG– 3’
● Blunt Ends: PvuII 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
0.8
● Observe fragmentation of DNA

Log Fragment Size (kbp)

0.7
● Plot migration distance (mm) of 0.6
standards vs. Log fragment size 0.5

● Use graph to find size of 0.4

fragments, see p. 192 0.3

0.2
Fragments: 17.5 mm, 22.0 mm 0.1
y = -0.0432x + 1.4906
R² = 0.997
5.42 kb, 3.47 kb 0.0
0 10 20 30 40
● Find total size of plasmids by Migration Distance (mm)
adding up the fragments
 Plasmid Maps
● Used to determine location of
EcoRI +
restriction enzyme sites on EcoRI HindIII HindIII Marker

plasmid 8kb

● Perform restriction enzyme 7kb

digest, run gel, measure 6kb

fragments:
5kb
● EcoRI: 3 kb, 5 kb
4kb
● HindIII: 2 kb, 6 kb
3kb
● EcoRI + HindIII:
2 kb, 1 kb, 5 kb
2kb
● Total Size of Plasmid: 8 kb 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
Plasmid X HindIII
● EcoRI + HindIII: 2 (8 kb) 2 kb
2kb, 1 kb, 5 kb
● Total Size of Plasmid: 8 kb

EcoRI
3 kb
 Plasmid Maps: Pop Quiz
HindIII
Digestion Fragment Size (bp) 0 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
 Plasmid Maps: Pop Quiz
HindIII
Digestion Fragment Size (bp) 0 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

BamHI
No single cuts in plasmid 2800-1800 = 1000 bp 1000 bp
Therefore, use 1st double cut 2800-1000 = 1800 bp
 Plasmid Maps: Pop Quiz
HindIII
Digestion Fragment Size (bp) 0 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

Use EcoRI + BamHI: BamHI

2800-2600 = 200 bp Should be directly next to EcoRI 1000 bp
BamHI site 1200 bp
2800-200 = 2600 bp
 Plasmid Maps: Pop Quiz
HindIII
Digestion Fragment Size (bp) 0 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

Check math with last double digest: BamHI

2800-1600 = 1200 bp EcoRI 1000 bp
2800-1200 = 1600 bp Plasmid Map complete! 1200 bp
 SP6 HindIII
Identifying Our Plasmids
pGEM3
● Using your restriction digest gel, REL

identify fragments from by size AmpR

● PvuII AhdI PvuII

● AhdI ORI BamHI
PvuII
● PvuII + AhdI
SP6 BamHI
PvuII
● How many fragments should you
pGEM4
have in each lane? REL
AmpR
● Identify which plasmid is which
by differences in size of two PvuII AhdI
sites HindIII
ORI T7
PvuII
Procedure: Chapter 6 – Week 2
● Restriction Enzyme Digest
● 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
● Restriction Enzyme Digest
● Prepare samples in 0.5 ml centrifuge tubes:
Single Digestions (x4) Double Digestions (x2)
1 µl 10 X Buffer 4 (NEBL) 1 µl 10 X Buffer 4 (NEBL)
2 µl plasmid DNA (~0.5 µg) 2 µl plasmid DNA (~0.5 µg)
6.5 µl Water (change with DNA) 6 µl Water (change with DNA)
0.5 µl PvuII or AhdI 0.5 µl of PvuII and AhdI
10 µl Total Volume 10 µl Total Volume
● Estimate DNA mass from agarose gel from week 1
● Vortex, Digest at 37°C for 1 hr
Procedure: Chapter 6 – Week 2
● Agarose Gel Electrophoresis
● Prepare Gel:
– While digest is running, pour 1% agarose gel (1 gel/ group)
● Sample Preparation:
Single Digestions X 4 Double Digestions X 2
2 µl 6X Sample Buffer 2 µl 6X Sample Buffer
10 µl of Single Digest 10 µl of Double Digest
12 µl Total Volume 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
● Agarose Gel Electrophoresis
● 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:
● Stain in ethidium bromide, 10 – 15 min
● De-stain in water, 1 min
● Image Gel:
– Take picture of agarose gel on gel dock