University:
Boston University
Course:
CAS BI 525 | Biology of Neurodegenerative Diseases
Academic year:

2021
Views:

250

Pages:

20
Author:

EverveNaxiq5h

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 pGEM4 from E. coli Rel Each contains: AmpR ● REL Gene Ahd I 3.57 ● SP6 Promoter ● Ampicillin resistance gene ● Origin of replication ● Restriction enzyme recognition sites You will need to identify which of your plasmids is pGEM3 & which is pGEM4 ● Include a labeled map with your lab report pGEM3-Rel 5.27 Kb Pvu II 1.92 ori Pvu II 2.50 SP6 promoter Pvu II 0.55 Rel pGEM4-Rel 5.27 Kb AmpR Ahd I 3.57 ori Pvu II 2.50 Overview of Mini‐Prep for Plasmid Isolation Need to separate nucleic acids from cell membranes and proteins ● Step 1: Cell lysis by detergents: SDS − ● 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 − ● Detergents will disrupt cell membrane and expels cytoplasm 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 for Plasmid Mini‐Prep Bacterial Cells Centrifuge, Lysis with NaOH and SDS Bacterial cytosol expelled into mini‐prep solution Treat with KOAc Detergent Precipitate Centrifuge Phenol / Chloroform Extraction Supernatant: Plasmid DNA, bacterial RNA, carbohydrates, proteins, lipids Bottom organic layer: Lipids Pellet: Unlysed cells, cell debris and attached chromosomal DNA Top aqueous layer: Plasmid DNA, RNA Wash nucleic acid pellet Resuspend with TE buffer Plasmid DNA Ethanol Precipitation Calculating DNA Concentration (spec) ● UV Absorbance ● Dilute 2 µl of mini‐prepped plasmid with 998 µl of TE buffer (1:500 dilution) ● Record A260 nm values from Cary‐60 specs ● These A260 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 – ● For DS DNA: 1.0 O.D. = 50 µg – ● 1.0 O.D. = Amount of nucleic acid that gives A260 = 1 in 1 ml 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 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 Supercoiled ladder (to measure size) Minnasota ladder (to measure mass) Visually removing bacterial RNAs -RNase S M A B +RNase A B • Bacterial RNAs are released into purification along with plasmid DNA • 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 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 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) 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 μL 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 μL 6X Sample Buffer 3 µL 3 μL 1 mg/ mL RNase (TF bench) ‐‐‐‐‐‐‐‐ 2 μL DI Water 15 µL – (Plasmid DNA µL) 13 μL – (Plasmid DNA μL) 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 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 – ● – Do not use more than 6 – 7 μl for UV‐absorbance exercise – Convert these O.D./mL measurements into mg/mL of DNA 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 – ● Adjust concentration as necessary so that 0.01 < A260 < 1.0 100 ng of Plasmid DNA in the band / 2 μl plasmid DNA in gel = 50 ng/µl Compare the results of the two methods in your post‐lab Chapter 6 Week 1: Before the lab period, you should have: Completed your prelab Title, date, introduction, procedures, and a flowchart of your plasmid isolation procedure Turned in your Chapter 5 post-lab write-up At the end of lab, you should have: Isolated your plasmid DNA (save DNA preps for week 2 and BI422) Ran your samples on an agarose gel Stained and imaged your gel

Chapter 6: Isolation of Plasmid DNA

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