Biology 475

Molecular Biology

Large-Scale Plasmid

Midi Prep

DNA Fingerprinting

National Science Foundation

Western Oregon University

Yellowstone National Park

The isolation of large amounts of plasmid DNA from insert-containing clones is necessary because subsequent DNA sequence analysis requires high levels of pure starting DNA. We will be using a moderately expensive plasmid isolation kit (Promega, Inc.) because it produces DNA compatible with our sequencing apparatus. As compared with the rapid mini-prep, large-scale plasmid isolation methods all share three basic parts: (1) a way to gently lyse the cellular hosts; (2) a way to crudely separate plasmid from the total cell extract; and (3) a way to purify and concentrate plasmid DNA.

In order to compare your samples using DNA fingerprinting methods - you will set up restriction digests based on some moderately tricky pre-lab analysis. Recall that restriction enzyme digest reactions require template DNA, enzyme(s), an appropriate enzyme buffer, and some water. Today, you will set up uncut samples, single digests, and double digests using interesting enzymes that should reveal more complex cutting patterns than those produced by EcoRI last time. As you will see, there are many challenges to this. For example, two enzymes we want to cut at the same time may function to lesser extents in non-ideal buffers. Charts are available in the form of company literature (in our case: Gibco BRL Life Technologies) to assist us when faced with the problem of making recipes and figuring these kinds of compatibility issues out. Additionally, you will analyze the enzyme's recognition sequence and calculate - based on probability - how many times each enzyme is predicted to cut the clone. Does the pattern you obtain match this prediction - why or why not?

Finally, you may have to contend with poor cutting as many interesting enzymes do not cut as well as EcoRI. For this reason, you will be preparing and running uncut plasmid DNA and have to contend with some new concepts. For example: although size is the primary factor that determines how far a given molecule will move through a simple agarose gel, conformation plays an equally important role. Well-prepared, uncut plasmid is "supercoiled" (tightly packed by proteins) and thus it moves farther than it would if not coiled. Poorly-prepared, uncut plasmid has additional lighter and slower bands, each of which corresponds to DNA that was damaged or nicked due to mishandling. In this way, the quality of a plasmid preparation (i.e. how much you beat it up) can often be deduced by examining uncut DNA. Most importantly, you can compare uncut plasmid to cut plasmid to assess for cutting problems. After setting up your digests (both cut and uncut), you will pour gels but we will load, run, stain, and photograph them for you… unless, of course, you are SO FAST that you can load them too!

Plasmid Isolation Procedures

The Day Before
Add ampicillin to 75-100 ml LB broth to a final concentration of 50 ug/ml.

Inoculate with one 16S rRNA clone; label appropriately. Each student will prepare plasmid from 1 or 2 clones - each of which represents insert-positive isolates from the mini-prep.

Incubate at 37 degrees C with shaking for at least 8-12 hours.

The Day Of
Pre-chill the centrifuge by turning on and setting to 4 degrees C. We will (hopefully) have remembered to do this. Ask us when you come into lab if we remembered.

Each student will receive 1 or 2 sterile round-bottom centrifuge tubes specific for our Sorvall rotor. Tubes hold about 35-40 ml. Decant half your culture into the tube; fill just to the point before the neck curves in.

Pellet cells at 10,000 rpm for 10 minutes at 4 degrees C.

Pour supernatant into collection beakers that contain bleach.

Add remaining 35-40 ml to existing pellet and re-pellet. Blot tubes upside down on a paper towel for 1 minute.

Add 3 ml Resuspension Buffer. DO NOT VORTEX because this will shear the genomic DNA into little pieces that will co-precipitate with the proteins later. The best way to resuspend is by gently pipetting up and down until the pellet disperses.

Add 3 ml Cell Lysis Solution and FIRMLY invert tube about four times. The suspension should begin clearing and turn into what looks and feels like egg white (let sit up to 5 minutes).

Add 3 ml Neutralization Solution and FIRMLY invert tube about four times. The solution should turn white and look powdery. Place on ice for five minutes.

Centrifuge at 12,000 rpm for 20-30 minutes. Be EXTREMELY gentle as you remove, carry, and handle the tubes following this step.

Carefully transfer supernatant to a sterile 50 ml conical tube by filtering through a cheesecloth square that has been folded once. There should be no solid in the transferred liquid.

Label a column tip with the clone number and secure it onto a vacuum port before proceeding. Make sure the port stopcock you are using is "off."

Our multi-port vacuum device was hand-designed and constructed by Danny. Be nice to it or Sarah and Danny will make you rebuild it.

Add 10 ml DNA-binding Resin (which has been evenly resuspended - i.e. shaken up well - prior to measuring) to your filtered crude lysate. Pour the resin/lysate mix into the column tip and apply a vacuum until the sample has been sucked through. The approximate time: 2-5 minutes.

After stopping the vacuum, add 20 ml Column Wash Solution and turn on the vacuum. Sucking the wash solution will take longer (10-15 minutes).

Start a water bath boiling - you will need it in about 10-15 minutes.

After the wash appears to have run through the column, run the vacuum an extra 30 seconds to "dry" before finally breaking the vacuum via your stopcock.

Using a serrated paring knife, cut and break the tip of the tip as shown in class (this is sort of like separating the lunar space module from the rocket, for lack of a better image).

Place the final tip into a 1.5 ml centrifuge tube, the cap of which you have cut off, and spin for 2 minutes in a microfuge at max speed. This step thoroughly removes residual wash solutions. Discard the centrifuge tube with the residual fluid and replace with a clean centrifuge tube; if you are careful, you can leave the lids on (i.e. lean them as though they were flying clockwise).

Add 300 ul of ultra-clean water that has been placed in the boiling water bath; wait one minute after adding the water to your column tip.

Centrifuge 20 seconds (ONLY) at max speed.

Remove the column tip and label the new tube using white lab tape and permanent ink. Update your label after quantitating the DNA. Proper labels indicate sample (site/clone number), date of preparation, DNA concentration, and your initials.

DNA Fingerprinting Pre-Lab

Recall your EcoRI recipe:

            DNA - 5 ul (this will be the same for your DNA)
            EcoRI (10 units/ul) - 0.3 ul (or 3 units)
            Buffer 3 (all buffers are 10X) - 1.5 ul
            Sterile Water - 8.2
            Total = 15 ul

Use this information PLUS company literature provided to set up the following recipes. YOU SHOULD HAVE THESE FINISHED when you walk into lab…

Background Table (will be part of your website)

Figure Out	HaeIII	HhaI	HindIII
Recognizes…
Bacterial Source
Best Buffer
Next Best Buffer
Units/ul
Cost
Reaction Temp.
# Cuts per 5 kb

Recipe Table (will be part of your website)

Recipe	DNA	Enzyme(s)	Buffer & Amt	Water
Uncut
HaeIII only
HhaI only
HindIII only
HhaI/HindIII