Molecular Biology Techniques: Myocardin & Cell Phenotype

• The lab focuses on micropipetting, restriction digestions, and DNA gel electrophoresis.
• The fundamental approach to investigate gene function involves altering its expression, achieved by introducing an exogenous DNA construct, using a plasmid or virus, into cells or tissue of interest.
• The workflow of a cell physiologist will be performed to characterize how gene over-expression impacts cellular phenotype.
• Analysis will be carried out focusing on changes in cellular appearance, localization of intracellular proteins and structures, and mRNA levels.
• The exogenous gene being expressed is called myocardin.
• It makes of use of a plasmid that expresses myocardin under the control of an EF1alpha promoter, ensuring consistent expression over several days, in addition to green fluorescent protein (GFP).
• This allows for visually confirming which cells take up and express the exogenous genes, focusing on the plasmid.
• Plasmids expressing myocardin and another for a different project were acquired.
• The labels on the plasmid samples fell off during transport and must be distinguished experimentally using restriction enzyme cutting site patterns.
• This week's goal is to confirm the correct plasmid for use.

Lab work plan

• Orientation to the lab and equipment/materials, find you lab partner.
• Review of lab safety protocols - covering any accidents, spills, showers, eye wash, hand washing, falls, sharps etc.
• Practice with simulation skills relating to micropipetting at the bench.
• Determination of the concentration of purified plasmid will be performed.
• Digestion of a small amount of each plasmid sample into fragments.
• Separation and visualization of fragments to identify each plasmid.
• Work with your lab partner/bench-mates/teaching team.
• If data collected are not what you expect, it can be an opportunity to troubleshoot your work.
• Problem-solving in research is more important than achieving the "right answer" on the first attempt.

Safety Considerations

• Be prepared, ask questions, and don't rush.
• Handling of SybrSafe® DNA-binding dye requires gloves.
• Handle gels as directed.

Experimental Aims:

• Design a restriction digestion experiment for the confirmation of the myocardin expression plasmid, also known as pMYOCD-IRES-GFP, or pMYOCD for short.

Learning Objectives:

• Learn micropipetting techniques
• Perform restriction digests of plasmid DNA
• Prepare/run DNA agarose gel electrophoresis.
• Link band pattern to plasmid map.
• Record-keeping and documentation of lab work practices

Protocol notes

• First: record your partner/team contact information
• Before lab: lab orientation which includes "Pipetting & Micropipetting" and videos
• Practice solid comfort level with this central skill because Pipetting is often required
• When holding a micropipette: Hook the pipette over your index finger of your dominant hand.
• Feel the plunger and tip ejector.
• The first stop is the "accurate" location for drawing up liquid.
• The second stop is for fully dispensing liquid, but not for drawing liquid, inaccuracy.
• Attach pipette tip firmly by pressing the tip into the box.
• Eject the tip by pressing the ejector button.

Micropipetting volumes

• Know the highest and lowest maximum volumes for your pipette
• Practice feeling the stops at each maximum volume
• Choose the right pipette, and pipette water or food coloring into a surface.
• Pipette three separate spots, each of 1000uL.
• Pipette three separate spots, each of 100uL.
• Pipette three separate spots, each of 9uL and maintain consistent size
• With a P20 set to 2uL, aspirate 2uL and pipette it all completely into a spot.
• A mistake would be to push the plunger to the 2nd stop before drawing up liquid, then pipette all this liquid into a spot.
• Compare sizes of spots - which is actually 2uL?

Making a solution:

• Prepare your own tube: 7.5uL blue + 22.5uL red + 275 uL yellow
• Check the colour and compare with the instructor.
• Set the P1000 to 305 uL and aspirate sample and ensure no drops or bubbles.
• Troubleshoot differences in results
• The Nanodrop Lite spectrophotometer determines DNA concentration and purity using very small volumes and captures an absorbance spectrum.
• The line may be long so set the gel cassette while you wait.

Working with Nanodrop

• If the spec has already been blanked, then you don't need to re-blank.
• The Nanodrop should first be “blanked" with a sample of dilute TE to baseline DNA-free absorbance spectrum and wipe off with kimwipe
• Load 1uL of sample, flick DNA tube, and click “measure.”
• Record the DNA concentration output as well as the A260/A280 ratio.
• Getting errors? Troubleshoot pipetting onto pedestal, and avoid bubbles.
• Part C deals with restriction Digests - where there are 2 plasmid samples
• pMYOCD-IRES-GFP, pCDNA3.1-NEAT1 are visualized using restriction enzymes.

Planning the enzymes experiment and choosing enzymes

• The enzymes create a unique pattern of band sizes that allows which plasmid is tested.
• Decide what restriction enzymes will work.
• Chose a plasmid with a clearly different set of bands for each of the two plasmids.
• The restriction enzymes available this semester are Sall, EcoRI, Ndel, Ncol, Dpnl, BamHI and ClaI.
• Your lab notebook, should note two enzyme(s) to use, also note the rationale for your choice.
• Fragments that are too small (<100bp) or too big (>9kbp) are difficult to resolve on a gel.
• Large fragments (>9kbp) may be difficult to distinguish from uncut plasmid.
• Calculate predicted sizes of fragments that you expect to generate from all of your restriction digest reactions.
• Each plasmid, will perform “double digest" reactions ("single digest” reactions and no-enzyme reactions).
• Organize your predictions according to the reactions in your lab book.
• Choose enzymes in partnership, other choices are ok.

Plasmid maps

• Features to notice - pUC ORI" or "ori" origin duplication; ampR ampicillin-resistance gene RE cut-sites in italics.

Planning the Restriction Digest Reactions:

• Write reaction mixtures in an incomplete (a.k.a. empty) table in your lab book and leave enzyme names blank.
• Each reaction mixture should be a total 20uL volume.
• The volume consist of buffer (stock is 10X), restriction enzyme(s) (1uL each), plasmid (~1ug), and water.
• Prepare an un-cut (no enzyme) reaction, and one or more single-cut reactions as controls.
• The amounts of plasmid and water to be added can be calculated if you know that the plasmid DNA concentration is 375ng/uL.
• Lab setting up Restriction Digest Reactions - Decide on one shared plan and finalize your tables with your partner.

Reaction mixtures and procedure

• Label a sterile 0.5 mL tube for each reaction.
• Prior to the experiment, talk about which components you should add first vs. last in parallel.
• Assemble reaction components in parallel and avoid contamination and clean the tip every time to pipette the enzyme.
• Vortex and tap down the tubes collect sample in the bottom.
• Incubate the reactions at 37°C for 15 minutes.
• Start preparing the gel to load.

Gel electrophoresis - separation and visualization digest fragments.

• The goal is to determine which plasmid is which and involves sharing a gel with another group.
• Prepare gel casting tray as shown and tape ends.
• The comb is placed at 15-well and placed in the tray.
• Weigh out 0.4 g of agarose powder in a flask and mix.
• Add 40 mL of 1X TBE buffer and swirl.
• Place flask in microwave and heat and swirl.
• Use gloves when dealing with heated flask.
• Let it cool slightly by swirling the flask use folded paper towels.
• Add DNA dye and swirl gently and pour mixture into the gel and top off with pipette tip.
• Let it set to cool completely and work on the next page.
• Plan the other groups, and right down in your notebooks the arrangement including 1 ladder.
• Each log into cloud account make folder and subfolder for partners to avoid plagiarism.

Pipetting into the gel

• What does a gel and its well feel like?
• Holding a pipette tip place in well and bottom is known
• Loading sample in well.
• Plant stabilize bench hands.
• Gently place tip into well
• Pipette and dispense
• Mix the sample is dispersed completely
• Try to avoid bubbles, push sample and re-draw
• After loading, look at see how it loaded.
• Gel loaded should now make mistake on purpose to what cause.

Loading and Running the gel

• Submerge the gel and remove the casting tray and place and tray with gel in the electrophoresis chamber.
• Fill the electrophoresis chamber to cover the gel by 1-2 mm and remove the well comb.
• Load ladder with the invitrogen
• Load 10micro litres to choose for ladder.
• Load 10micro litres directly when.
• Avoid bubbles that cause uneven bands.
• The voltage should set to 200 volts, and watch tracking dye separate.
• Is your gel is what is there you are to find
• Then tidy up everything to find
• Use accruis for smart dock gel with Canvas.
• Steps handle tray gently use buffer dipose it, and use label waste

Followed by following for the picture set

• Align gel platform, turn on blue and turn light know bands
• place and the blue light on know your smartphone take or enlarge.
• Talk team image resolution the run with make time
• look at with image
• use email and share quickly
• Place gels, and put into Safe
• Run used to run again
• ice can leave tube.
• Tips use dispose
• clean with ethanol on disposal bags.
• Results image brigth compress
• Annoations of bands.
• Check bands for size.
• note and preddict.
• TAE buffer can volume up to 1%