micro 329 lecture 5

MICR 329

• Exercise 5, Parts 1 and 2 relates to restriction digestion and gel electrophoresis

Pipetting

• Hold the pipette in a near-vertical position when preparing to use it
• Depress the plunger smoothly to the first stop
• Immerse pipette tip in liquid when aspirating
• Allow the plunger to return to rest position smoothly
• Wait one second for all the liquid to transfer into the tip
• Place the pipette tip at a 10- to 45-degree angle against the inside wall of the receiving vessel while dispensing
• Depress the plunger smoothly to the first stop position
• Wait one second, then depress the plunger to the second stop position (purge)
• Slide the pipette tip up on side to remove the tip from the vessel wall

Materials for Restriction Digest

• Remove DNA from the freezer; Genomic, Plasmid, Hind III, Sal I, buffers, lambda DNA
• Use pipettes and tips
• Use 1.5mL centrifuge tubes

Restriction Digest Protocol

• Confirm calculations
• Set up digest
• Digest lambda genomic material with HindIII
• Digest A.fischeri gDNA with SalI
• Lecture occurs when incubation takes place

Bioluminescent E. coli

• Can be created from V. fischeri genomic DNA
• It is digested with restriction enzymes and combined with a plasmid
• Join with DNA ligase, resulting in a recombinant plasmid
• Then transformed into E. coli

How to Get Target Gene into Vector

• Both Target gene and Plasmid "cut" with restriction enzymes
• DNA ligase used to join pieces, resulting in recombinant plasmid

pGEM®-3Zf(+) Vectors features

• Vectors are 3,197bp
• Has multiple cloning sites to insert new genes

Anatomy of an Expression Plasmid

• Plasmids require three main things:
• Origin of replication (Ori): Where DNA replication of plasmid originates
• Multiple cloning sites (MCS): AKA polyclonal site or polylinker, Used for engineering new plasmids and inserting genes
• Selectable trait: Usually antibiotic resistance gene (ampicillin, chloramphenicol, etc)
• Restriction enzymes “cut” the plasmid at Multiple cloning sites (MCS)
• MCS have different cut sites for different restriction enzymes

Restriction Enzymes

• Endonucleases cut DNA
• Type II is used in research
• Cuts unmethylated DNA
• Cuts specific sites
• In nature serves as bacterial immune system against phages
• Bacteria methylate their own DNA to prevent cutting

Type II Endonucleases

• Requires Mg2+ as a cofactor
• EDTA (metal cation chelator) was added in the cell extraction buffer to prevent the activity of these enzymes cleaving the DNA during isolation
• One reason why we have a special buffer
• Does not require ATP
• Cuts at short, palindromic sequence
• Reading the sequence 5' to 3' is the same as the sequence on the other strand of DNA read 5' to 3'
• Results in blunt or sticky ends (AKA overhangs, cohesive ends)

Blunt vs Sticky Ends

• Blunt: All enzyme cuts are compatible with each other, but may ligate other non-desired products and the gene(s) can't be ligated in correct orientation
• An example is Smal from Serratia marcescens
• Sticky: Not all enzymes cuts are compatible with each other, rarely ligate non-desired products, and can easily choose the direction of gene(s) ligated in
• An example is Sall from Streptomyces albus G

Confirming Restriction Digestion with Gel Electrophoresis

• Handling Ethidium Bromide
• Prepare and cast gels
• Prepare samples: Part B Steps 11-13
• Load Gels

DNA Methylation and Restriction Protocols

• Brief Lecture on Handling Ethidium Bromide
• Prepare and cast gels
• Return where gels cool in the hood
• Prepare samples Part B Steps 11-13
• Go get your gels and load them
• Lecture in the last 45 mins

DNA Methylation and Restriction.

• Safety
• Pipettes and Tips
• Gel chambers (group drawers)
• Digest from last class (leave in the freezer for now)

Ethidium Bromide (EtBr)

• Is a MUTAGEN.
• Binds ssDNA, dsDNA, and RNA
• Action: intercalates between stacked bases
• Leads to mutations during DNA replication
• This changes the shape of DNA slightly, which allows mismatching of bases during DNA replication, leading to mutations
• EtBr does not change the sequence of the DNA on the gel
• It is not a good idea to transform an organism with stained DNA

Ethidium Bromide Safety

• Must use:
• Fume hood
• Lab coat (fully buttoned)
• Pants
• Closed toed shoes
• Gloves
• Allow gel to cool (No steam) before adding EtBr
• Let gel solidify in the hood
• Keep the open bottle in the hood at all times
• Handling: (hazardous materials in general)
• Try not to pick up an open bottle
• Put the lid on to pass it
• Keep it low to the table when you pipette
• Screw on the lid when you are not actively pipetting it
• Work slowly and in the moment
• Keep the Hood sash low (I'll show you)
• Always transfer using a secondary container!

Casting Technique

• Pour slowly
• Avoid bubbles
• Let them settle before you pour
• Pop them with a pipette tip if they make it in
• Common errors include:
• Use the right buffer and must match running buffer, because high voltage can also cause this
• Use a clean flask

Before Casting the Gel

• Remove your DNA from the Freezer, put it on ice, and leave on your benchtop
• Bring:
• 250mL Flask
• Gel tray (just the tray)
• Comb (6-tooth)
• Lab coat and notebook

After Returning to the Gel

• Consider the purpose of the gel and each tube
• Add loading dye to the aliquots to be run on the gel
• Prepare Loading samples:
• Perform part B, steps 11-13 of gel electrophoresis protocol
• Start with Step 13 for a feel for pipetting the loading dye (share an aliquot if needed)
• Only add dye to the tubes indicated (double check labels!)
• Collect solidified Gel from 508 (use a secondary container)
• Prepare Gel Chamber (step 8-10)
• Load Gel with samples (step 15), because the gel is SOFT it is best if you are confident in technique
• Run gel (16-18).
• Check the voltage and Wires
• Sketch a mock gel layout, because we will fill this in with expected Lane patterns

Structure of DNA

• In gel electrophoresis, DNA is "linearizing" the circular DNA
• A single cut opens the circle, Bacterial genomes and plasmids are circular
• Tubes D and F are the samples and the rest are controls
• Tube B is a reference ladder to tell if digestions were successful
• Lambda genomic material will be digested with HindIII and A. fischeri gDNA with Sall
• Digest lambda phage to make ladder

Ladder Production

• A phage to make ladder
• Digest gDNA to get lux operon
• Sall sites are located on either side of lux operon in A. fischeri
• Sall site is in the MCS of pGEM
• Gel electrophoresis allows visualization of cuts that can be done to see if experiments were successful

Restriction Modification and DNA Methylation

• Restriction enzymes used to cut DNA
• Escherichia coli (and other bacteria) methylates(modifies) its DNA at specific, short DNA sequences
• Adenines (A) with Dam methylase enzyme
• Cytosines (C) with Dcm methylase enzyme
• Other enzymes exist which depends on species and strain of bacteria
• DNA is methylated, E. coli recognizes it as "self”
• "Non- self" unmethylated DNA from phages is targeted by E. coli's restriction endonucleases and degraded
• Methyl groups protect DNA from these endonucleases
• Some restriction endonucleases CAN degrade methylated DNA
• Try to cut A. fischeri gDNA and E. coli plasmid DNA with the endonuclease Sall, due to the capability of cutting Dam and Dcm methylated DNA

Restriction activity of Type II endonucleases to engineer DNA

• Did we methylate our DNA? How do we know?
• What cofactor is required to cut DNA?
• Is ATP required for this reaction?
• What type of sequences are targeted by restriction enzymes?
• What type of ends are produced after restriction?

Review of digested lambda genomic material from HindIII and A. fischeri gDNA with Sall

• Lambda phage is digested to make a ladder
• Aims of gDNA digestion:
• to excise the lux operon, so that it can be put in a plasmid
• Sall sites are located on either side of lux operon in A. fischeri
• Visualize cuts using gel electrphoresis whether it can be successful or not

Electrophoresis

• The movement of charged particles in response to an electric field
• Agarose gel electrophoresis is useful for DNA/RNA (>25 bp long)
• Other types in research include:
• SDS-PAGE, Useful for proteins & small DNA/RNA (e.g., 6 bp fragments)
• Capillary electrophoresis, and DNA sequencing

Agarose Gel Electrophoresis

• The size of DNA fragments depends on speed of DNA migration
• 5 kb = big/heavy and slow
• 50 bp = lightweight and fast
• Agarose concentration
• More agarose = higher density = smaller pore size = increased friction = slower migration
• Voltage
• High voltage = fast migration
• Structure of DNA also impacts
• Speed of DNA impacts movement in during electrophoresis

Electrophoresis Buffers

• TAE,TBE: only subtle differences
• Roughly 50 mM Tris (T) as final concentration
• pH should be 7.5-7.8
• Buffers (TBE) are used in:
• The apparatus itself (liquid cover the gel completely)
• The agarose gel itself (solid)
• Critical to maintain the same ionic strength between the gel and the solution that surrounds it

Loading Dye

• Loading dye has multiple functions during electrophoresis
• Usage:
• Typically kept as a 6x stock
• Mixed with the DNA sample right before loading into gel
• Components help in 3 ways:
• Glycerol or Sucrose (sugar): Keeps sample in the well
• Sticks to the DNA, weighing down the sample, making it sink
• Bromophenol Blue: A rough marker, migrates independent of DNA sample; runs with 250-500 bp, helps you see your sample, and easier to load a blue-dyed sample into wells

DNA and RNA Visualization

• DNA and RNA must be stained to be visualized with Ethidium Bromide You can stain DNA on a gel with two methods:
• In-gel staining: Dye is to molten agarose before polymerization occurs (aka hardening)
• And as the DNA sample migrates, it picks up dye
• Some positive results include: quick! Immediately able to observe DNA and image immediately after running, as well as chemical waste is solid, so easier to dispose of safely.
• Negative results include: dye influences on DNA migration, ever so slightly
• Post-staining: Agarose gel is soaked in a dye bath in the absence of light
• In your tupperware, add stain to TAE for 30-45 mins (after DNA has run).
• Some positive results include: cheaper over time, less chance of contamination (handle tubes less), can be done if one forgets to add dye to gel, and sharper bands
• Negative results include: takes longer
• Binds ssDNA, dsDNA, and RNA
• Action: intercalates between stacked bases; weaker binding to ssDNA
• Under UV light, gives off fluorescence
• 590 nm light re-emitted == orange/red color
• This is NOT the same a bioluminescence

Next Steps

• Dephosphorylation of plasmid and Ligation takes place in the next lab
• Needs to be done by Wednesday
• Do ligation calculations ahead of time
• Need concentration of Digestion gDNA and plasmid (calculate from restriction protocol)
• Readings and Quiz
• Topic Paper Choice due 2/21/25