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 kcat from your post-lab write-up

data; check blackboard for a guide

• Chapter 6AB begins Wednesday, Nov 20th and ends

next Tuesday, Nov 26th

• Your chapter 6AB post-lab write-up is due during the last week
• f 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 4th

• 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

• We are isolating pGEM3 and

pGEM4 from E. coli

• Each contains:
• REL Gene
• 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

Maps are on p. 205 of the Lab Manual AmpR AmpR

• ri
• ri

Rel Rel

SP6 promoter SP6 promoter

Ahd I 3.57 Ahd I 3.57 Pvu II 0.55 Pvu II 1.92 Pvu II 2.50 Pvu II 2.50 pGEM3-Rel 5.27 Kb pGEM4-Rel 5.27 Kb

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)

Bacterial Cells Bacterial cytosol expelled into mini‐prep solution Detergent Precipitate Supernatant: Plasmid DNA, bacterial RNA, carbohydrates, proteins, lipids Bottom organic layer: Lipids Top aqueous layer: Plasmid DNA, RNA Wash nucleic acid pellet Plasmid DNA Pellet: Unlysed cells, cell debris and attached chromosomal DNA

Treat with KOAc Centrifuge Phenol / Chloroform Extraction Ethanol Precipitation Resuspend with TE buffer

Flow Chart for Plasmid Mini‐Prep

Centrifuge, Lysis with NaOH and SDS

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

– 1.0 O.D. = Amount of nucleic acid that gives A260 = 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

S M A B A B

• RNase +RNase
• 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

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 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

–

– 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

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

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

– Adjust concentration as necessary so that 0.01 < A260 < 1.0

– 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

– 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