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) SP6 promoter ● You have isolated plasmid "A" and Rel "B" from E. coli pGEM3-Rel 5.27 Kb AmpR ● Each contains: REL Gene Ahd I 3.57 ● Pvu II 1.92 SP6 Promoter ● ori Pvu II 2.50 Ampicillin resistance gene ● Origin of replication SP6 promoter ● Pvu II 0.55 Restriction enzyme recognition sites ● ● You will need to identify which of Rel your plasmids is pGEM3 & which is pGEM4-Rel 5.27 Kb pGEM4 AmpR Include a labeled map with your lab ● Ahd I 3.57 report ori Pvu II 2.50 Maps are on p. 199 of the Lab Manual

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 Eco RI: Yields products with 5’ overhangs that can base pair Eco RV in complex with with each other DNA (1RVC) Phosphodiester 5’ –GAATTC– 3’ 5’ –G ‐OH ‐2 O 3 PO‐ AATTC– 3’ Bond Cleavage 3’ –CTTAAG– 5’ 3’ –CTTAA‐ OPO 3 HO‐ G– 5’ 2‐

What are the products of a restriction enzyme digest? ● Digest DNA with restriction 1.0 enzymes 0.9 ● Run gel and observe 0.8 Log Fragment Size (kbp) fragmentation of DNA 0.7 0.6 ● Plot migration distance (mm) of 0.5 standards vs. Log fragment size 0.4 ● Use graph to find size of 0.3 fragments, see p. 192 0.2 y = ‐0.0432x + 1.4906 Fragments: 17.5 mm, 22.0 mm 0.1 R² = 0.997 0.0 5.42 kb, 3.47 kb 0 10 20 30 40 ● Find total size of plasmids by Migration Distance (mm) adding up the fragments

Restriction Maps ● Used to determine location of Eco RI + restriction enzyme sites on Eco RI Hind III Marker Hind III plasmid 8kb ● Perform restriction enzyme 7kb digest, run gel, measure 6kb fragments: 5kb ● Eco RI: 8 kb 4kb ● Hind III: 1 kb, 7 kb 3kb ● Eco RI + Hind III: 1 kb, 2 kb, 5 kb 2kb ● Total Size of Plasmid: 8 kb 1kb

Restriction Maps ● Used to determine location of Eco RI restriction enzyme sites on plasmid 0 kb (8 kb) ● Perform restriction enzyme digest, run gel, measure fragments: ● Eco RI: 8 kb ● Hind III: 1 kb, 7 kb Plasmid X Hind III ● Eco RI + Hind III: 2 2 kb (8 kb) 2kb, 1 kb, 5 kb ● Total Size of Plasmid: 8 kb Hind III 3 kb

SP6 promoter Identifying your Plasmids Rel ● Using your restriction digest gel, pGEM3-Rel identify fragments from by size 5.27 Kb AmpR ● Pvu II Ahd I 3.57 Pvu II 1.92 ● Ahd I ori Pvu II 2.50 ● Pvu II + Ahd I SP6 promoter Pvu II 0.55 ● How many fragments should you Rel have in each lane? pGEM4-Rel 5.27 Kb ● Identify which plasmid is which AmpR by differences in size of two Pvu II Ahd I 3.57 sites ori Pvu II 2.50

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 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 Pvu II or Ahd I 0.5 µl of Pvu II and 0.5 µl Ahd I 10 µl Total Volume 10 µl Total Volume ● 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 37 o 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) – You’ll need a minimum of 7 wells. Use the comb with 10 teeth ● Sample Preparation: For all reactions: 2 µl 6X sample buffer 10 µl Restriction digest rxn 12 µl Total Volume ● Load Gel: – 6 samples and 1 standard / gel – Standard: Linear DNA Minnesota Molecular (Table II, p. 187)

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