Announcements Last week of labs Chapter 6C Chapter 6AB postlab - - PowerPoint PPT Presentation

Announcements

• Last week of labs – Chapter 6C
• Chapter 6AB post‐lab write‐ups are due in lab
• Chapter 6C in‐lab worksheet is turned in before the

end of lab

• Last 15 minutes of today’s discussion section will be

for online student evaluations

Chapter 6: Restriction Mapping

Purpose of Week 2: A) Create a restriction map using restriction enzymes on your plasmids B) Determine between plasmid A & B which is pGEM3‐Rel and pGEM4‐Rel

 pGEM3‐Rel (reverse orientation of ORF)  pGEM4‐Rel (ORF in correct orientation)

Your plasmids (review)

• You have isolated plasmid "A" and

"B" from E. coli

• Each contains:
• REL Gene
• SP6 Promoter
• Ampicillin resistance gene
• Origin of replication
• Restriction enzyme recognition sites
• You will need to identify which of

your plasmids is pGEM3 & which is pGEM4

• Include a labeled map with your lab

report

Maps are on p. 199 of the Lab Manual AmpR AmpR

• ri
• ri

Rel Rel

SP6 promoter SP6 promoter

Ahd I 3.57 Ahd I 3.57 Pvu II 0.55 Pvu II 1.92 Pvu II 2.50 Pvu II 2.50 pGEM3-Rel 5.27 Kb pGEM4-Rel 5.27 Kb

Restriction Enzymes

• Restriction Endonucleases
• Recognize and cleave DNA to make smaller fragments
• DNA fragments can be cloned into new molecule using DNA

ligases

• Genomic DNA 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’

• verhangs 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)

What are the products of a restriction enzyme digest?

• Digest DNA with restriction

enzymes

• Run gel and 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 10 20 30 40 Log Fragment Size (kbp) Migration Distance (mm)

Restriction Maps

• Used to determine location of

restriction enzyme sites on plasmid

• Perform restriction enzyme

digest, run gel, measure fragments:

• EcoRI: 8 kb
• HindIII: 1 kb, 7 kb
• EcoRI + HindIII:

1 kb, 2 kb, 5 kb

• Total Size of Plasmid: 8 kb

EcoRI HindIII EcoRI + HindIII Marker 1kb 2kb 3kb 4kb 5kb 6kb 7kb 8kb

Restriction Maps

• Used to determine location of

restriction enzyme sites on plasmid

• Perform restriction enzyme digest,

run gel, measure fragments:

• EcoRI: 8 kb
• HindIII: 1 kb, 7 kb
• EcoRI + HindIII: 2

2kb, 1 kb, 5 kb

• Total Size of Plasmid: 8 kb

EcoRI 0 kb (8 kb) HindIII 2 kb HindIII 3 kb

Plasmid X (8 kb)

Identifying your 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

AmpR AmpR

• ri
• ri

Rel Rel

SP6 promoter SP6 promoter

Ahd I 3.57 Ahd I 3.57 Pvu II 0.55 Pvu II 1.92 Pvu II 2.50 Pvu II 2.50 pGEM3-Rel 5.27 Kb pGEM4-Rel 5.27 Kb

Chapter 6C Procedure

Workflow for Chapter 6 week 2:

• Calculate volumes for restriction digests (done in prelab)
• Prepare restriction digest reactions
• Cast a 1% agarose gel
• Prepare samples and gel tank
• Load samples and run gel
• Stain, destain, and image gel on UV-gel doc

If you are taking Biochemistry 2, you will save your plasmids for Lab 8 next semester!

Procedure: Chapter 6 – Week 2

Restriction Enzyme Digest – Do these calculations before coming to lab

• Calculate plasmid DNA concentration from week 1 gel
• Calculate volume of plasmid DNA needed for ~0.5 µg for each reaction
• Each reaction should total 10 µl in volume

‐ Adjust DI water amount if necessary ‐ Do not change buffer or enzyme volume amounts

• Incubate reaction at 37oC for 1 hr

Single Digestions (2 per plasmid) Double Digestions (1 per plasmid) 1 µl 10 X Cut Smart Buffer 1 µl 10 X Cut Smart Buffer 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 0.5 µl 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) – You’ll need a minimum of 7 wells. Use the comb with 10 teeth

• Sample Preparation:
• Load Gel:

– 6 samples and 1 standard / gel – Standard: Linear DNA Minnesota Molecular (Table II, p. 187)

For all reactions: 2 µl 6X sample buffer 10 µl Restriction digest rxn 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 dye reaches bottom 1/5 of gel – Record volts and running time in your lab notebook

• Staining and De‐staining of Gel:
• Stain in ethidium bromide, 10 min
• De‐stain in water, 5 min
• Image Gel:

– Take picture of agarose gel on gel dock

Chapter 6 Week 2

Before the lab period, you should have:

Completed your prelab Title, date, introduction, procedures Calculations for restriction digest reactions

At the end of lab, you should have:

Digested and ran your reactions on your 1% agarose gel Stained and destained your gels Taken a picture of your restriction digest gel on the gel-dock