Announcements Chapter 5 post-lab write-ups are due at the beginning - PowerPoint PPT Presentation

Announcements • Chapter 5 post-lab write-ups are due at the beginning of your Chapter 6AB lab • Various ways for calculating k cat from your post-lab write-up data; check blackboard for a guide • Chapter 6AB begins Wednesday, Nov 20 th and ends next Tuesday, Nov 26 th • Your chapter 6AB post-lab write-up is due during the last week of labs after Thanksgiving Break (due during Chapter 6C) • This is first half of your Chapter 6 post-lab grade • Chapter 6C will resume after Thanksgiving on Wednesday, Dec 4 th • Chapter 6C post-lab write-up will be an in-lab worksheet completed during your Chapter 6C lab • The second half of your Chapter 6 post-lab grade

Chapter 6: Isolation of Plasmid DNA Purpose of Week 1: A) Isolate plasmid DNA from E. coli B) Determine DNA concentration by two methods  UV absorbance (Cary ‐ 60 UV/Vis)  Gel electrophoresis (agarose gels)

Our Plasmids SP6 promoter ● We are isolating pGEM3 and Rel pGEM4 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 ● Rel ● You will need to identify which of 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. 205 of the Lab Manual

Overview of Mini ‐ Prep for Plasmid Isolation Need to separate nucleic acids from cell membranes and proteins Step 1: Cell lysis by detergents: SDS ● Detergents will disrupt cell membrane and expels cytoplasm − Step 2: Addition of potassium acetate: Precipitates detergents and high molecular ● weight impurities Detergents & membrane debris will be pelleted from nucleic acids and proteins − Step 3: Extraction with Phenol / Chloroform: Removes proteins ● Separates nucleic acids in an aqueous layer from the lipids in the organic layer − and denatured, precipitated proteins at the interface Step 4: Precipitation of Nucleic Acids using 100% ethanol ● Nucleic acids (DNA & RNA) are precipitated and pelleted out of solution − Steps 1 – 4 will allow you to generate mini ‐ prep plasmid DNA samples Step 5: Digest RNA using RNase during sample prep for gel electrophoresis ● RNA is enzymatically digested, but will still contribute to 260 nm absorbance − values (keep this in mind during this week’s lab)

Flow Chart Centrifuge, Lysis Bacterial Cells with NaOH and SDS for Plasmid Bacterial cytosol expelled Mini ‐ Prep into mini ‐ prep solution Treat with KOAc Detergent Precipitate Centrifuge Phenol / Supernatant: Plasmid DNA, bacterial Pellet: Unlysed cells, cell debris RNA, carbohydrates, proteins, lipids and attached chromosomal DNA Chloroform Extraction Bottom organic layer: Lipids Top aqueous layer: Plasmid DNA, RNA Ethanol Precipitation Wash nucleic acid pellet Resuspend with Plasmid DNA TE buffer

Calculating DNA Concentration (spec) ● UV Absorbance Dilute 2 µl of mini ‐ prepped plasmid with 998 µl of TE buffer (1:500 dilution) ● ● Record A 260 nm values from Cary ‐ 60 specs These A 260 nm values are direct readings for the units of O.D./mL ● These initial O.D./mL values reflect the diluted sample in 1 mL ● Back ‐ calculate to the concentrated mini ‐ prep sample you isolated by multiplying by ● the sample prep dilution factor ( i.e. , multiply by 500 for this example) In post ‐ lab, calculate [DNA] from O.D. – optical density units – 1.0 O.D. = Amount of nucleic acid that gives A 260 = 1 in 1 ml ● For DS DNA : 1.0 O.D. = 50 µg – 20 O.D./mg DNA ● For RNA: 1.0 O.D. = 40 µg – 25 O.D./mg RNA

Agarose Gel Electrophoresis ● Relative DNA migration rates depends on: ● Size and conformation (supercoiled versus closed circular) ● Concentration of agarose in the gel ● Applied voltage ● Your gel will melt if it gets too hot! ● All DNA has the same charge ‐ to ‐ mass ratio with a negative charge ● Your negatively charged DNA will migrate toward the positively charged red wire ( cathode )

Ethidium Bromide Staining • Ethidium Bromide (EtBr) is an interchalating agent Be very careful when handling EtBr as it is a potential carcinogen • Will fluoresce under UV light when bound to nucleic acids From Wikimedia commons

Observing Plasmid DNA ● An Ethidium Bromide stain is used to observe DNA ● Multiple forms of Plasmid DNA: ● Supercoiled circular DNA ● Nicked circular DNA ● Linear DNA ● Our system’s migration pattern: ● Nicked circular slowest ● Linear ● Supercoiled fastest http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/supercoils.jpg

Calculating DNA Concentration (gel estimation) ● Agarose Gel ● Each gel will have two markers: ● Supercoiled ladder – to measure size of supercoiled DNA sample ● Minnesota ladder – to measure mass of supercoiled DNA sample ● For measuring DNA concentrations: ● Compare your sample’s signal intensity with a band in the Minnesota ladder ● Estimate the mass of DNA in your sample ● Divide your sample’s mass by the volume of DNA used in the corresponding lane ● For example: 30 ng of estimated mass divided by 2 µl of DNA used in sample = 15 ng/µl Look at marker tables in p. 192

Supercoiled ladder (to measure size) Minnasota ladder (to measure mass) Look at marker tables in p. 192

Visually removing bacterial RNAs • Bacterial RNAs are released into -RNase +RNase purification along with plasmid DNA S M A B A B • 260 nm wavelength on the UV/Vis detects the nucleobases, which are present in both DNA and RNA • Your UV/Vis-based DNA concentrations will be significantly greater than your gel-based estimations • We will also remove E. coli RNAs in week 2 to visualize smaller digested DNA fragments S = supercoiled ladder M = Minnesota ladder A = plasmid A B = plasmid B

Chapter 6AB Procedure Workflow for Chapter 6 week 1: • Isolate plasmid DNA • Cast a 1% agarose gel • Measure nucleic acid concentration with UV-Vis spec • Prepare samples and gel tank • Load samples and run gel • Stain, destain, and image gel on UV-gel doc Make sure to save your plasmids for week 2! If you are taking Biochemistry 2, you will save your plasmids for Lab 8 next semester!

Procedure: Chapter 6 – Week 1 Lysing E. coli cells ● Get 2 aliquots of cells transformed with Plasmid A & B ● In week two, you will use restriction enzymes to determine which is pGEM3 ‐ Rel and pGEM4 ‐ Rel ● Centrifuge 1 min – remove supernatant ● Add 100 μ l of GTE and vortex to resuspend cell pellet ● Add 200 μ l of NaOH/SDS lysis solution, mix by inversion and ice for 5 min ( do not lyse for more than 5 min ) ● Add 150 μ l of potassium acetate solution, mix by inversion ● Centrifuge for 5 min at top speed – pipette supernatant into a clean eppendorf tube, discard pellet

Procedure: Chapter 6 – Week 1 ● Plasmid Mini ‐ Prep ● Add 1:1 phenol:chloroform (v/v) to your samples ● Pull from the bottom layer of the stock bottle – Phenol is highly toxic, can cause severe burns and throat irritation – This step MUST be done in the hood! ● Vortex/shake your samples vigorously for 30 sec ● Centrifuge to separate aqueous and organic phase ● Transfer top aqueous phase to clean labeled tube ● Discard bottom layer and all phenol:chloroform waste directly in the hood

Procedure: Chapter 6 – Week 1 ● Plasmid Mini ‐ Prep: ● Add cold 100% ethanol to aqueous layer, mix well ● Centrifuge ~15 ‐ 30 minutes ● Remove supernatant, wash pellet with cold 70% ethanol ● Centrifuge 1 ‐ 2 minutes ● Remove supernatant and air dry – be careful not to remove pellet at the same time ● Add 35 μ L TE , vortex/pipet to dissolve pellet ● Final sample = mini ‐ prepped plasmid DNA

Procedure: Chapter 6 – Week 1 ● Agarose Gel Electrophoresis – TF's will demo in lab ● Prepare Gel: – Prepare casting tray using gel box walls – Pour into casting tray, add comb, and let solidify

New Procedure: Chapter 6 – Week 1 ● Agarose Gel Electrophoresis ● Sample Preparation: For Each Plasmid A & B, set up the following Sample No RNase RNase digest Plasmid DNA: 0.1 – 0.2 OD units 2 ‐ 7 µL 2 ‐ 7 μ L 6X Sample Buffer 3 µL 3 μ L 1 mg/ mL RNase (TF bench) ‐‐‐‐‐‐‐‐ 2 μ L – DI Water 15 µL – (Plasmid DNA µL) 13 μ L – (Plasmid DNA μ L) Total Volume 18 µL 18 µL – You will generate a total of 4 sample preps in your group – Incubate your samples at 37 ° C for 10 min to digest the RNA before loading onto your gel ● Load Gel: – Run gel with another group: 8 samples + 2 standards/ gel – 2 Standards for each gel: See table p. 192 ● Supercoiled DNA Marker ● DNA Mass, Minnesota Molecular

Procedure: Chapter 6 – Week 1 ● Agarose Gel Electrophoresis ● Run Gel: – What is the charge on DNA? Which direction will it run? – Run gel at 100 ‐ 150 V until dyes separate – If you run the gel faster it will MELT! ● Staining and De ‐ staining of Gel: – Stain in ethidium bromide, 10 – 15 min – Ethidium Bromide is a known carcinogen/mutagen! – Use gloves and dispose of waste properly! – De ‐ stain in water, 2 min ● Image Gel: – Take picture of agarose gel on gel dock