Announcements Labs resume this Wednesday (11/29 ) with Chapter 6AB - PowerPoint PPT Presentation

Announcements • Labs resume this Wednesday (11/29 ) with Chapter 6AB • Chapter 5 post‐lab write‐ups are due at the beginning of your Chapter 6AB lab • Blackboard announcement and material for k cat calculation • Your chapter 6AB post‐lab write‐up is due during the last week of labs (Chapter 6C) • The first half of your Chapter 6 post‐lab grade • 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  Gel electrophoresis

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 ● ● 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

Mini‐Prep for Plasmid DNA 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 − ● Step 5: Digest RNA using RNase 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 + RNase

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

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

DNA Concentrations ● UV Absorbance ● Dilute 2 µl of mini‐prepped plasmid with 998 µl of TE buffer ● Record A 260 nm ● Calculate [DNA] from OD – optical density units – 1.0 OD = Amount of nucleic acid that gives A 260 = 1 in 1 ml ● For DS DNA : 1.0 OD = 50 µg – 20 OD/mg DNA ● For RNA: 1.0 OD = 40 µg – 25 OD/mg RNA

DNA Concentrations ● 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. 187

Supercoiled ladder (to measure size) Minnasota ladder (to measure mass) 15 ng 15 ng 25 ng 30 ng 40 ng 75 ng 50 ng 60 ng 15 ng 30 ng 10 ng 20 ng 15 ng 10 ng 10 ng Look at marker tables in p. 187

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 10‐20 sec – remove supernatant ● Add 100 μl of GTE and vortex to resuspend cell pellet ● Add 200 μl of NaOH/SDS lysis solution, mix by inversion and ice for 5 min ( do not lyse for more than 5 min ) ● Add 150 μl 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) – vortex vigorously for 30 sec – Phenol is highly toxic, can cause severe burns and throat irritation – This step MUST be done in the hood! ● 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 – be careful not to remove pellet at the same time ● Add 35 μL TE + RNase , vortex to dissolve pellet ● Incubate at 37 ° C, 10 min – to digest RNA ● 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

Procedure: Chapter 6 – Week 1 ● Agarose Gel Electrophoresis ● Sample Preparation: For Each Plasmid Sample Volume Plasmid DNA: 0.1 – 0.2 OD units 2‐7 µL 6X Sample Buffer 1.7 µL DI Water (8.3 µL) – (Plasmid DNA µL) Total Volume 10 µL ● Load Gel: – Run gel with another group: 4 samples + 2 standards/ gel – 2 Standards for each gel: See table p. 184 ● 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, 5 min ● Image Gel: – Take picture of agarose gel on gel dock

Procedure: Chapter 6 – Week 1 ● Determination of DNA Concentration ● Before running gel – Measure concentration by UV‐ absorbance – Dilute 2 μl of DNA to 1.0 ml in TE buffer – Adjust concentration as necessary so that 0.01 < A 260 < 1.0 – Do not use more than 6 – 7 μl for UV‐absorbance ● After gel is complete – Calculate concentration from gel – Use DNA mass standard and estimate relative intensity of each band – Divide this amount by the amount of DNA in the sample – 100 ng of Plasmid DNA in the band / 2 μl plasmid DNA in gel = 50 ng/µl ● Compare the results of the two methods

Chapter 6 Week 1: Before the lab period, you should have:  Completed your prelab  Title, date, introduction, procedures  Turned in your Chapter 5 lab notebook At the end of lab, you should have:  Isolated your plasmid DNA ( save aliquots for week 2 and Biochem II )  Ran your samples on an agarose gel  Stained and imaged your gel