Announcements Lab Exam 12/9 12/11 during discussion ~20 mul9ple choice ques9ons Will require a calculator All Chapter 6 Labs Due by 12 noon on Wed. 12/12 Chapter 6ab Discussion Dates Lab Dates Lab Due Dates Tuesday lab on 12/3 Chapter 6c 12/3 12/5 12/4 12/10 All Sec9ons Noon, 12/12 Boxes outside SCI 162 Lab Exam 12/9 12/11 Chapter 6: Week 2 Restric;on Digest of Plasmid DNA Purpose: 1) Learn about restric9on enzymes and plasmid maps 2) Perform restric9on enzyme digest to iden9fy your plasmids Restric9on Enzymes Restric;on Endonucleases Recognize and cleave DNA to make smaller fragments DNA fragments can be cloned into new molecule using DNA ligases Genomic DNA oden protected from diges;on in the cell by DNA methyla;on 3 Types of Restric;on Enzymes: Type I: Cleave DNA at random sites, > 100 from restric9on sequence, requires ATP Type II: Cleave DNA within recogni9on sequence, does not require ATP Type III: Cleave DNA about 25 bp from recogni9on sequence, requires ATP Type II: Restric9on Enzymes Only cut DNA at specific recogni9on sequences Recogni9on sequences typically 4-6 bp long O\en 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) 5 G- OH - 2 O 3 PO- AATTC 3 3 CTTAA- OPO 3 HO- G 5 1
Type II: Restric9on Enzymes Restric9on Enzymes can give: Type II: Restric9on Enzymes Restric9on Enzymes can give: 5 Overhangs: EcoRI 3 Overhangs: PstI 5 GAATTC 3 3 CTTAAG 5 5 CTGCAG 3 3 GACGTC 5 Overhangs are oden called S;cky Ends 5 Overhangs: EcoRI 3 Overhangs: PstI 5 G- OH - 2 O 3 PO- AATTC 3 3 CTTAA- OPO 3 HO- G 5 5 CTGCA- OH - 2 O 3 PO- G 3 3 G- OPO 3 HO- ACGTC 5 Blunt Ends: 5 CAGCTG 3 3 GTCGAC 5 Blunt Ends: 5 CAG- OH - 2 OPO 3 - CTG 3 3 GTC- OPO 3 HO- GAC 5 What are the products of a restric9on enzyme digest? Digest DNA with RE Run gel Observe fragmenta9on of DNA Plot migra9on 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) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 y = - 0.0432x + 1.4906 0.1 R² = 0.99702 0.0 0 10 20 30 40 Migra;on Distance (mm) Used to determine loca9on of restric9on enzyme sites on plasmid Perform restric9on enzyme digest, run gel, measure fragments: EcoRI: 3 kb, 5 kb : 2 kb, 6 kb EcoRI + : 2 kb, 1 kb, 5 kb Total Size of Plasmid: 8 kb Plasmid Maps EcoRI EcoRI + Marker 8kb 7kb 6kb 5kb 4kb 3kb 2kb 1kb 2
Digestion of Known Plasmid 246 EcoRI 845 ApaBI Total bp = 5547 Single digest ApaBI Single digest EcoRI Double digest ApaBI + EcoRI Plasmid Maps Used to determine loca9on of restric9on enzyme sites on plasmid Perform restric9on enzyme digest, run gel, measure fragments: EcoRI: 3 kb, 5 kb : 2 kb, 6 kb EcoRI + : 2 2kb, 1 kb, 5 kb Total Size of Plasmid: 8 kb, EcoRI 0 kb (8 kb) Plasmid X (8 kb) EcoRI 3 kb 2 kb 2800 2800 + 1800, 1000 + EcoRI 1600, 1200 EcoRI + 2600, 200 2800 2800 + 1800, 1000 + EcoRI 1600, 1200 EcoRI + 2600, 200 No single cuts in plasmid Therefore, use 1 st double cut 2800-1800 = 100 2800-1000 = 180 100 3
2800 2800 + 1800, 1000 + EcoRI 1600, 1200 EcoRI + 2600, 200 2800 2800 + 1800, 1000 + EcoRI 1600, 1200 EcoRI + 2600, 200 Use EcoRI + : 2800-2600 = 20 2800-200 = 260 Should be directly next to site EcoRI 100 120 Check math with last double digest: 2800-1600 = 120 2800-1200 = 160 Plasmid Map complete! EcoRI 100 120 Iden9fying Our Plasmids Using your restric9on digest gel, iden9fy fragments from by size AhdI + AhdI How many fragments should you have in each lane? Iden9fy which plasmid is which by differences in size of two sites Amp R Amp R AhdI AhdI ORI ORI SP6 pgem3 SP6 pgem4 REL T7 REL Restric9on Enzyme Digest If you are taking Biochemistry 2, make sure to label and save your plasmids for next semester! 4
Restric9on Enzyme Digest Prepare samples in 0.5 ml centrifuge tubes: Single s (x4) Double s (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 or AhdI 0.5 µl of and AhdI 10 µl Total Volume 10 µl Total Volume Es9mate DNA mass from agarose gel from week 1 Vortex, Digest at 37 C for 1 hr Prepare Gel: While digest is running, pour 1% agarose gel (1 gel/ group) Sample Prepara;on: Single s X 4 Double s 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) Run Gel: What is charge on DNA? Which direc9on will it run? Run gel at 100-125 V un9l dyes separate and are near bosom of gel Record volts, amps, running 9me, 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 5