University:
Boston University
Course:
CAS BI 107 | Biology 1
Academic year:

2023
Views:

251

Pages:

17
Author:

Kelsey Bennett

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 ● 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) ● 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: ● ● ● ● 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 ● 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: ● Restriction Enzyme Digest ● Agarose Gel Electrophoresis ● Restriction Enzyme Digest ● 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 ● Vortex, Digest at 37°C for 1 hr ● Agarose Gel Electrophoresis ● 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) ● 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

Restriction Digest of Plasmid DNA

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