Announcements. Chapter 5 Labs Due this week! Lab Exam 12/10 12/12. Discussion Dates Lab Dates Lab Due Dates

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1 Announcements Chapter 5 Labs Due this week! Lab Exam 12/10 12/12 Discussion Dates Lab Dates Lab Due Dates Chapter 5 11/28 12/3 in lab Chapter 6ab 11/26 11/28 11/28 12/3 Chapter 6c 12/3 12/5 12/5 12/10 All Sections Noon, 12/12 Boxes outside SCI 162 Lab Exam 12/10 12/12

2 Chapter 6: Week 1 Isolation of Plasmid DNA Purpose: 1) Learn about plasmid DNA 2) Isolate plasmid DNA 3) Determine DNA concentrations from gel electrophoresis and UV absorbance

3 Plasmids Independent, circular, self-replicating form of DNA Encode for genes separately from chromosomes Typically found in bacteria Must contain origin of replication (ORI) Must contain a selectable marker such as antibiotic resistance gene Typically contain several restriction enzyme binding sites Often used for cloning inserting particular gene into bacterial genome

4 Our Plasmids SP6 HindIII We are isolating pgem3 and pgem4 Each contains: Amp R pgem3 REL REL Gene SP6 Promoter Ampicillin resistance gene Origin of replication Several restriction enzyme sites You will need to identify which of your plasmids is which Amp R AhdI ORI PvuII BamHI PvuII SP6 BamHI PvuII pgem4 REL Include a labeled map with your lab report AhdI Maps are on p. 194 of the Lab Manual ORI PvuII HindIII T7

5 Mini-Prep for Plasmid DNA Isolation Need to separate nucleic acids from cell membranes and proteins Need to further separate DNA from RNA Step 1: Cell lysis by detergents: SDS or Triton X-100 Step 2: Addition of potassium acetate: Precipitates detergents and high molecular weight impurities Step 3: Extraction with Phenol / Chloroform: Removes proteins Step 4: Precipitation of Nucleic Acids by 100% ethanol Step 5: Separation of DNA from RNA by RNase

6 Agarose Gel Electrophoresis DNA separates due to: Size and conformation Concentration of agarose in the gel Applied voltage All DNA is negatively charged no effect on separation Multiple forms of Plasmid DNA may be found in gel: Closed circular DNA Supercoiled Nicked circular DNA Linear DNA The buffer system you use determines which runs fastest Our system: Supercoiled > Linear > Nicked circular

7 Agarose Gel Electrophoresis Rate the DNA travels through a gel is inversely proportional to Log MW (kb) Can increase rate by increasing the voltage However, increasing voltage will also increase heat in the system Your gel may melt if it gets too hot! Better separation typically occurs at lower voltage

8 DNA Concentrations Agarose Gel UV Absorbance Look at the bands in your linear standard Each has mass assigned to it Estimate relative intensities of your sample compared to standard Multiply sample mass by amount of each sample in gel DNA Size DNA Mass 60 ng 15 ng 30 ng 10 ng 20 ng 15 ng Dilute plasmid to 2 µl/1 ml 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 Look at marker table p. 184

9 Flow Chart for Plasmid Mini-Prep Phenol / Chloroform Extraction Treat with KOAc Bottom organic layer: Proteins Centrifuge, Lysis with NaOH and SDS Centrifuge Supernatant: Low molecular nucleic acids, proteins Chromosomal DNA, ribosomes Detergent Precipitate Bacterial Cells Top aqueous layer: Low molecular weight nucleic acids Low molecular weight nucleic acids RNA, some proteins, small DNA Pellet: SDS, High molecular weight debris Ethanol Precipitation Treat with RNase Plasmid DNA

10 Procedure: Chapter 6 Week 1 Plasmid Mini-Prep Agarose Gel Electrophoresis Determination of DNA Concentration 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!

11 Procedure: Chapter 6 Week 1 Plasmid Mini-Prep Get 2 aliquots of cells containing 2 different plasmids Centrifuge sec remove supernatant Vortex to resuspend pellet in buffer Lyse cells with NaOH and SDS, mix gently by inversion Treat with KOAc, mix by inversion / shaking Centrifuge 5 min decant supernatant, discard pellet

12 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 phases, 1 min Remove top aqueous phase to clean labeled tube Discard bottom layer and all phenol : chloroform waste directly in the hood

13 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 TE + Rnase, vortex to dissolve pellet (p. 178 Change!) Incubate at 37 C, 10 min remove RNA Final Sample = Pure Plasmid DNA

14 Procedure: Chapter 6 Week 1 Agarose Gel Electrophoresis Prepare Gel: Prepare casting tray using gel box walls Pour into casting tray, add comb, and let solidify

15 Procedure: Chapter 6 Week 1 Agarose Gel Electrophoresis Sample Preparation: For Each Plasmid Load Gel: Sample Volume Plasmid DNA: OD units 2-7 µl 6X Sample Buffer 1.7 µl DI Water 8.3 Plasmid DNA µl Total Volume 10 µl 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

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

17 Procedure: Chapter 6 Week 1 Determination of DNA Concentration Before running gel Measure concentration by UVabsorbance Dilute 2 μl of DNA to 1.0 ml in TE buffer Adjust concentration as necessary: 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