warm-up november 28, 2007

Warm-Up November 28, 2007

Are you currently applying or have you already applied to

college? If yes, where? If no, what do you plan to do when

you graduate?

Agenda

• Warm-up/ take attendance

• Did you finish the quick strawberry DNA lab writing? If not, do it now!

• Be sure to turn in yellow page for QUICK DNA lab before you leave

• Lecture on chemicals and procedures used in DNA extractions- fun fun fun!

Homework

• Pre-lab for Friday’s lab

• Bring fruit or vegetable if you want to purify its DNA

Purifying DNA, the long way

• Measure 10.00 g of plant parts into a weigh boat.

• Don’t drop things on the balance, gently place them.

• Be sure to tare the balance after putting on the weigh boat.

Place the plant part and 3 mL deionized water into a cold mortar

and pestle.

• Mortar and pestles are in the fridge- help yourself

• Mix with dI water:• So that we can have a solution

for plant guts to go into. We want dI water b/c we don’t want to introduce things we don’t know about.

• grind plant parts until smooth:• to make sure the cell walls are

destroyed and the cell’s guts are released into the solution

• micropipettor: push, stick, suck• graduated pipette: keep

vertical• mortar and pestle: Ms

America- elbow elbow wrist wrist; no clanging, no bashing; we should not hear any loud noises when working with a mortar and pestle

• Cold slows down enzymes that want to chew up the DNA

With a clean 1 ‑ mL pipette, add 1 mL of 50% dish washing detergent

solution/ lysis buffer.

• Detergent solution: to break apart membranes; SDS is hydrophobic and hydrophilic just like membranes are; plasma and nuclear membranes

• Hold the bottle so you can see the tip and where it is going. Watch the liquid go in the graduated pipette or micropipettor.

http://www.moleculeoftheday.com/images/sds.gif

Polar head

Non-polar tail

(12 carbons)

Orientations of SDS or other fatty acid molecules that have a polar

head and non-polar tail

http://www.moleculeoftheday.com/2006/05/04/sodium-dodecyl-sulfate-for-that-fresh-no-scum-feeling/

Micelle- laundry Bilayer similar to cell membranes

Add 0.5 mL 10 mM EDTA

• enzymes that destroy DNA use divalent cations;

• bacteria that could contaminate the solution also use divalent cations to break apart DNA

• Actually EDTA is not added to DNA purifications that will be used for PCR. PCR is dependent on Mg+2 concentrations and EDTA will bind the Mg+2 and therefore will interfere with the lab.

Structure of EDTA

Disodium EDTA

This diagram does not mention that an antibiotic hasbeen added, too, but it has.

Apparently Tris-EDTAhelps neutralize acidic bodyparts such that antibiotics will workbetter with Tris-EDTA.

From a Veterinarian’s website:http://www.dermapet.com/articles/new_concepts.html#anti

Use a second clean 1‑mL pipette to add 1 mL of 200% contact lens

cleaner solution with papain • papain: protease- ase

means it is an enzyme; works on proteins; helps degrade proteins that could interfere with the purification; may break down histones so that DNA can even be more released

• Please be patient because the papain solution is made at the beginning of the lab. Don’t take more than 1 mL because we will run out.

Pour or scrape the plant mush into a clean small beaker. Label beaker

using tape and a sharpie.

• Beakers are in buckets

• Sharpies are in your lab drawer/ filing cabinet

• Tape is near the microwave (I hope)

• Don’t worry about beaker size- if you need a larger beaker, then use a larger beaker.

• Scraping…hmm… we’ll have to put something in the bucket for that.

Incubate the plant mixture in a 65 C water bath for 10 minutes

• denatures proteins- want to denature enzymes that can attack the DNA

• also allows membranes to loosen up

• Don’t stick your fingers in the beaker.

• If the beaker is too hot to handle, we may have a small problem- I don’t know if the school has beaker tongs- will have to look into that.

Transfer the beaker from the hot water bath to the ice bath for 5

minutes to stop the lysis

• to stop the denaturing process

• to encourage DNA to hydrogen bond back together if it did start to fall apart.

• Make an ice bath by putting ice in one of your larger beakers. You can get ice from the freezer unless it is already out on a table.

Filter and squeeze the plant mixture through 4 layers of

premoistened cheesecloth into a beaker.

• filter through cheesecloth: separate chunks from liquid

• we don’t want the chunks b/c our DNA is in the liquid- chunks will get in the way of future use of the DNA

• Wet the cheesecloth with water (tap water is ok, distilled water is better)

• Twist from above. This will remove solid plant material.

Pour the filtrate equally into two centrifuge tubes

• check that weight of each tube is within 0.5 g

• Our centrifuges only have one speed- just use the speed that it is set at. If it has more than one speed, ask Ms. Getz what to do.

• Unbalanced centrifuges can break. The rotor can break.

Imagine a picture of a centrifuge tube here- after the spin

http://fig.cox.miami.edu/~cmallery/255/255hist/ecbxp5x4x2.jpg

Pour the supernatant into a clean plastic tube.

• Flip back to previous slide to show supernatant and pellet

Slowly pipette or pour 1.5x volume of ice‑cold 95% ethanol carefully down the side of the tube so that

there are two phases. • ethanol displaces the

water and pushes the DNA out of solution

• Pour down the side so that the layers form.

At this point we’ll leave the tubes in the refrigerator so that the DNA can

go into the ethanol solution overnight.

• Be careful to not disturb the layering- this is one place where serious messing up can happen

Spool the DNA

• put the DNA into a 1.5 mL tube by scraping it off the glass rod against the lip of the tube.

• We hook the DNA to remove the DNA from the rest of the junk.

• don’t drop your tubes or jostle them before you hook the DNA

How to get a 1.5 mL tube out of the sterile beaker.

Monday!

Now we’re going to use the Quick DNA too

• Get your quick DNA microfuge tube from the refrigerator

Spin both tubes in a microfuge

• Make sure they are balanced!

• You may have to add a little more alcohol to one of the microfuge tubes to balance them.

• That is ok.

• You are KEEPING this pellet- it is the DNA.

Remove as much alcohol as possible from the DNA with a

micropipette or transfer pipette.

• ethanol will interfere with future labs so the DNA needs to be resuspended in TE unless we’re using the DNA for PCR (which we are not doing with the plant DNA)

Why does the DNA precipitate in the ethanol solution?

• DNA is less soluble in ethanol than in water; the ethanol pretty much slides in between water and DNA- the DNA will fold up on itself and float out of solution.

• The ethanol displaces “air” trapped in the water which is why you’ll see bubbles rise up with the DNA.

• Don’t drink the ethanol- it has stuff in it that will make you very sick.

Add approximately 0.5 mL TE buffer to resuspend the pellet.

• Why is TE buffer used?

• buffers- to keep the pH constant

• Tris- buffer

• EDTA- preservative