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 with each other

5’ –GAATTC– 3’ 3’ –CTTAAG– 5’

Phosphodiester Bond Cleavage

5’ –G-OH -2O3PO-AATTC– 3’ 3’ –CTTAA-OPO3

2- HO-G– 5’

EcoRV in complex with DNA (1RVC)

Type II: Restriction Enzymes ● Restriction Enzymes can give:

● 5’ Overhangs: EcoRI

● 3’ Overhangs: PstI

● Blunt Ends: PvuII

5’ –GAATTC– 3’ 3’ –CTTAAG– 5’

5’ –CAGCTG– 3’ 3’ –GTCGAC– 5’

5’ –CTGCAG– 3’ 3’ –GACGTC– 5’

Overhangs are often called

“Sticky Ends”

Type II: Restriction Enzymes ● Restriction Enzymes can give:

● 5’ Overhangs: EcoRI

● 3’ Overhangs: PstI

● Blunt Ends: PvuII

5’ –G-OH -2O3PO-AATTC– 3’ 3’ –CTTAA-OPO3

2- HO-G– 5’

5’ –CAG-OH -2OPO3-CTG– 3’ 3’ –GTC-OPO3

2- HO-GAC– 5’

5’ –CTGCA-OH -2O3PO-G– 3’ 3’ –G-OPO3

2- HO-ACGTC– 5’

What are the products of a restriction enzyme digest?

● Digest DNA with RE

● Run gel

● Observe fragmentation of DNA

● 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

y = -0.0432x + 1.4906 R² = 0.997

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 10 20 30 40

Log

Frag

me

nt

Size

(kb

p)

Migration Distance (mm)

Plasmid Maps

● Used to determine location of restriction enzyme sites on plasmid

● Perform restriction enzyme digest, run gel, measure fragments:

● EcoRI: 3 kb, 5 kb

● HindIII: 2 kb, 6 kb

● EcoRI + HindIII: 2 kb, 1 kb, 5 kb

● Total Size of Plasmid: 8 kb

EcoRI HindIII EcoRI + HindIII Marker

1kb

2kb

3kb

4kb

5kb

6kb

7kb

8kb

Plasmid Maps

● Used to determine location of restriction enzyme sites on plasmid

● Perform restriction enzyme digest, run gel, measure fragments:

● EcoRI: 3 kb, 5 kb

● HindIII: 2 kb, 6 kb

● EcoRI + HindIII: 2 2kb, 1 kb, 5 kb

● Total Size of Plasmid: 8 kb

HindIII, EcoRI 0 kb (8 kb)

HindIII 2 kb

EcoRI 3 kb

Plasmid X (8 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

Construct the restriction enzyme map for this plasmid

HindIII 0 bp

BamHI 1000 bp 2800-1800 = 1000 bp

2800-1000 = 1800 bp No single cuts in plasmid Therefore, use 1st double cut

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

BamHI 1000 bp Should be directly next to

BamHI site

EcoRI 1200 bp

Use EcoRI + BamHI: 2800-2600 = 200 bp 2800-200 = 2600 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

Construct the restriction enzyme map for this plasmid

HindIII 0 bp

BamHI 1000 bp EcoRI

1200 bp Plasmid Map complete!

Check math with last double digest: 2800-1600 = 1200 bp 2800-1200 = 1600 bp

HindIII SP6

REL

PvuII

BamHI

PvuII

ORI

AhdI

AmpR

pGEM3

Identifying Our Plasmids ● Using your restriction digest gel,

identify fragments from by size

● PvuII

● AhdI

● PvuII + AhdI

● How many fragments should you have in each lane?

● Identify which plasmid is which by differences in size of two PvuII sites

pGEM4

PvuII T7

HindIII

REL

PvuII

BamHI SP6

AmpR

AhdI

ORI

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:

● Estimate DNA mass from agarose gel from week 1

● Vortex, Digest at 37°C for 1 hr

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

Procedure: Chapter 6 – Week 2 ● Agarose Gel Electrophoresis

● Prepare Gel:

– While digest is running, pour 1% agarose gel (1 gel/ group)

● Sample Preparation:

● Load Gel:

– 6 samples and 1 standard / gel

– Standard: Linear DNA Minnesota Molecular (Table II, p. 184)

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

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