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) Phosphodiester -2 5’ – GAATTC– 3’ 5’ –G-OH O3PO-AATTC– 3’ 3’ –CTTAAG– 5’ Bond Cleavage 3’ –CTTAA-OPO 2- HO-G– 5’ 3 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 -2O PO-AATTC– 3’ ● 3 5’ Overhangs: EcoRI 2- 3’ –CTTAA-OPO3 HO-G– 5’ -2 5’ –CTGCA-OH O3PO-G– 3’ ● 3’ Overhangs: PstI 2- 3’ –G-OPO3 HO-ACGTC– 5’ -2 5’ –CAG-OH OPO3-CTG– 3’ ● Blunt Ends: PvuII 2- 3’ –GTC-OPO3 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 0.7 ● Plot migration distance (mm) of 0.6 standards vs. Log fragment size 0.5 0.4 ● Use graph to find size of fragments, see p. 192 0.3 Log Fragment Size (kbp) Size Fragment Log 0.2 Fragments: 17.5 mm, 22.0 mm y = -0.0432x + 1.4906 0.1 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 + EcoRI restriction enzyme sites on HindIII HindIII Marker plasmid 8kb 7kb ● Perform restriction enzyme 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 1200 bp 2800-200 = 2600 bp BamHI site 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 Identifying Our Plasmids SP6 HindIII 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 have in each lane? pGEM4 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 .