BISC411

                                EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL
 

                                       Restriction Enzyme Digestions and Agarose Gels

Restriction Enzyme Digestion:

Today you will be use the plasmid preparations that you generated in week 2 and the plasmid you generated in week 3 also. This will be a total of 3 plasmids being used.

We have discussed how the restriction enzyme digestions can inform us about which plasmid is which. You will be using the patterns generated to identify the plasmids. You will be told which buffers and enzymes to use with each plasmid being examined. Below are the conditions for the digestions:

 Appropriate digestion buffer (2X concentration): 10 microliters
 BSA:       1 microliter
 DNA (2 micrograms):     X microliters
 Enzyme:      1 microliter
 Water:       X microliters

Note: The volume of DNA is determined by the concentration of your samples, determined in the previous weeks.

Note: The final total volume of each digest should total 20 microliters.

Note: The BSA is often optional. When present, it improves the specificity of digestions for many enzymes.

The digests are vortexed briefly, spun down very briefly, and incubated at 37oC for 1 hour at least. During this time, you will make the agarose gels and prepare the molecular weight markers.

Upon completion of the digestions, you will add 4 microliters of the Blue-Juice dye buffer to each digest, vortex briefly, spin down briefly, and heat all samples, including the markers, at 65oC for 2 minutes, putting the samples on ice immediately upon completion of the heating.

Briefly spin down the samples and immediately load all of the sample into the wells of the agarose gels for analysis.
 

Agarose Gel Electrophoresis

While your DNAs are digesting, prepare an agarose gel as follows:

Prepare 1 liter of 1X running buffer (10X TEA) by diluting the 10X stock buffer down 1 in 10 with deionized water.

Weigh out 1 gram of agarose. Put it into the 250 ml. erlenmeyer flask.

Add 100 ml of the 1X buffer to the flask. Swirl gently. This will NOT dissolve at this point.

Cover the flask loosely with plastic wrap.

Microwave the mixture until the agarose has melted into the solution and it is completely clear. (Observe carefully. You can interrupt the run to check on things and restart it as needed. Do not let the agarose solution spill out or froth over the top of the flask. Remember the flask will be very hot – wear insulated gloves or use a thick wad of paper towels to move the flask.

Put the flask aside and allow it to cool at room temperature for about 10-15 minutes.

Place the gel tray into the apparatus in the pouring orientation as demonstrated. Place the well-forming comb into the tray.

After 10 minutes, test the temperature of your solution by attempting to touch the side of your flask to your cheek. If it stings, the solution is still too hot. Wait 5 more minutes and try again. It should feel quite warm but not painfully so. Obviously, do not leave a very hot flask on your skin for any length of time. We do not want any burns!

Add 10 microliters of ethidium bromide solution to the agarose solution carefully. Swirl gently being sure the deep red ethidium bromide dissolves uniformly. You will see the solution look at little bit pink. BE CAREFUL. Ethidium bromide is a mutagen. Never work with it without gloves. Discard the pipette tip you use to add it in the biohazard bag.

Immediately pour the solution into the gel tray. Pour slowly but not so slow that the solution begins to thicken. Look for any bubbles and burst them by pricking them with a pipet tip. Very tiny bubbles can be moved to the edges of the gel.

Move the well-former up and down once or twice to dislodge any possible bubbles trapped under the teeth. Again, do it carefully but do not allow the gel to thicken before you have finished this.

Allow the gel to harden at room temperature. Do not disturb it.

After about 20 minutes, the gel is hardened.

Carefully pull out the well former, using an upward pull to avoid disturbing the wells in the gel.

Manipulate the tray out of the apparatus and reposition it for loading and running. It fits snugly so you will need to apply some force to get it out but not so much that you smash up your gel. Remember, the DNA will move from negative pole to positive pole so position the wells accordingly.

Carefully pour enough 1X buffer nto the apparatus to completely cover the gel surface.

After loading the samples,  put the lid on the apparatus, connect the power, and run the DNA on the gel at 80V, constant voltage.

Continue this run until the dye front has moved half way down the gel (about 2 hours).

Shut off the power and carefully remove the tray and gel. Bring the gel to the gel documentation system and put the entire thing (still in the tray) onto the UV box. You will then be assisted in working the documentation system and obtaining a picture of your results.

You will measure the distance travelled by the molecular weight markers and, using the molecular weights provided by the instructor, construct a standard curve using semi-log graph paper. Graph the distance travelled on the normal x-axis and the size on the log scale y axis. This is done because the distance travelled is inversely proportional to the log of the molecular weight. Use this to determine the sizes of the digested DNA in your samples.