ERICKSON_P12:

RESTRICTION ENZYME DIGESTIONS

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride concentration in the reaction varies for different enzymes, and a set of standard buffers containing three concentrations of sodium chloride are prepared and used when necessary.. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC. See map for your assigned gene for a listing of restriction sites present.

1. Prepare the reaction for restriction digestion by adding the following reagents to a microcentrifuge tube in the order listed: 

               
               10X assay buffer          1.5 ul 
               DNA                              8 ul (0.5-2 ug)
                 10X BSA (1 mg/ml)     1.5 ul
                 sterile ddH20                q.s. to 15 ul ("q.s."= quantity sufficient)
               restriction enzymes*     0.5 ul each (usually 1-10 units per ug DNA)
                               Total volume 15 ul
 

*If desired, more than one enzyme can be included in the digest if both enzymes are active in the same buffer and the same incubation temperature. Refer to the vendor's catalog for the chart of enzyme activity in a range of salt concentrations to choose the appropriate assay buffer. Restriction enzymes are purchased from Promega, New England Biolabs,  Roche, Takara many others.

2. Gently mix by slowly pipetting up/down.  A 5 second spin is sometimes helpful.  Incubate the reaction at the appropriate temperature (typically 37 deg C) for 1-12 hours. 

3. Stop digest by adding Loading buffer to 1X final concentration. You can store the digest in the freezer at this point if convenient.  An aliquot of the digestion (about 1/2 of the digest reaction) should be assayed by agarose gel electrophoresis versus non-digested DNA and a size marker.

Agarose gel electrophoresis is employed to check a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in Tris-acetate-EDTA (TAE) buffer by heating in a microwave and the warm gel solution then is poured into a mold, which is fitted with a well-forming comb. The percentage of agarose in the gel varies. Although 1.0 % agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1.5 or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 100 volts for 0.5-1 hour at room temperature, depending on the desired separation. Size markers are co-electrophoresed with DNA samples, when appropriate for fragment size determination.  After electrophoresis, the gel is placed on a UV light box and a picture of the fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid camera.

Protocol

1. Prepare an agarose gel using a small gel box (1 % agarose gel: 35 ml of TAE buffer + 0.35 g of agarose) by combining the agarose and 1 X TAE buffer in a 250 ml Ehrlenmeyer flask, and heating in a microwave for 1-3 minutes until the agarose is dissolved (no chunks seen when swirled); repeat heating if necessary.

2. Add ethidium bromide (EtBr; 10 mg/ml stock) to a final concentration of 0.5 ug/ml; swirl or stir on stir plate the flask to mix, and pour the gel onto the cast with casting combs in place. Allow 10-20 minutes for solidification. CAUTION: Ethidium bromide is a mutagen and a possible carcinogen. Handle solutions only when wearing latex or vinyl gloves. 

3. Carefully remove the gel casting combs and place the gel in a horizontal electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs until the buffer just covers the agarose gel.

4. Samples containing DNA mixed with loading buffer are then pipetted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the anode, which is usually colored red (remember: Run to the Red). Electrophorese the gel at 100 V until the required separation has been achieved, usually 0.5-1 hour. Visualize the DNA fragments on a UV light box and photograph with a camera attached to a computer.  Print out results and paste into your lab notebook.

NOTES

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.

Additionally, several factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to advantage in optimizing separation of DNA fragments. Chief among these factors are:

Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.

Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.

If subcloning is your next step: Run the remaining portion of the restriction digests on a new gel.  Visualize DNA fragments using a long-wave length UV light source to protect DNA (our gel box is short UV); cut out desired fragments and store in separate microfuge tubes.  Elute DNA from gel using Qiagen spin columns according to Qiagen’s instructions.

Ethidium Bromide

Ethidium Bromide (EB) is commonly used as a non-radioactive marker for identifying and visualizing nucleic acid bands in electrophoresis and in other methods of gel-based nucleic acid separation. EB is a dark red, crystalline, non-volatile solid, moderately soluble in water, which fluoresces readily with a reddish-brown color when exposed to ultraviolet light (UV). Its formula is 2,7,-Diamino-10-ethyl-9-phenyl-phenanthridium bromide, CAS# 1239-45-8. Although it is an effective tool, its hazardous properties require special safe handling and disposal procedures.

Hazards
EB is a potent mutagen and moderately toxic after an acute exposure. EB can be absorbed through skin, so it is important to avoid any direct contact with the chemical. EB is also an irritant to the skin, eyes, mouth, and upper respiratory tract. It should be stored away from strong oxidizing agents in a cool, dry place, and the container must be kept undamaged and tightly closed.

Safety Precautions
People using EB should follow several safety procedures. The laboratory's Chemical Hygiene Plan should reference this Fact Sheet, which outlines safe handling of EB and proper cleanup procedures. EB users should receive documented safety training on its hazards. EB must appear on the laboratory's chemical inventory, with accurate estimates of on-hand and yearly use quantities. Pure EB should only be handled in a fume hood, with the user wearing protective equipment that includes a lab coat, closed-toe shoes, chemically resistant gloves, and chemical safety goggles (not just safety glasses).

Nitrile is an effective barrier to short-term exposure to EB. Gloves, such as Best Manufacturing's N-DEX® or others made of 100% nitrile, are available from most laboratory supply distributors. EB users should wash their hands after removing their gloves, even if they are certain the gloves weren't punctured.

An emergency eyewash and shower should be accessible nearby. Like all other toxics, EB should be used in a specially designated area where no eating or drinking is allowed. When using ultraviolet light to visualize EB, the user must wear UV-blocking eyewear or work in a UV cabinet with shielding glass in place.
Emergency Exposure Procedures
If EB contacts the eyes, immediately flush them with copious amounts of cold water for at least 15 minutes. (If it is available, an emergency eyewash is the best and safest way to do this.) For skin contact, immediately wash the affected area with soap and copious amounts of cold or cool water. If a person inhales EB dust, move him to an area where he can breathe fresh air. After any exposure to EB (via skin, inhalation, or eye contact), the affected person should immediately seek a medical evaluation from Tang Center or from Alta Bates Hospital if the exposure occurs while Tang Center is closed.

Spill Procedures
 Large Spill
Notify all others in the room that the spill has occurred. Evacuate the room or immediate area and call for assistance with the cleanup. Post the room with signage warning others of the spill, and prevent unnecessary entry into the area until the EH&S response team arrives. Provide any assistance and information you can to the spill responders.

Small Spill
 Use UV light to locate the spill; EB's fluorescence is easy to see. If the spill is powder, carefully wipe it up with wet paper towels and follow the decontamination procedure below. If the EB spill is liquid, absorb freestanding liquid with dry paper towels. Use a UV light source and illuminate the area to locate any remaining EB. Then use the decontamination procedure. After the decontamination procedure, re-survey the area with UV light to ensure that all the EB has been collected.

Disposal
Unwanted solid EB, gels, and all working solutions must be disposed of properly. Gels should be allowed to dry by evaporation prior to disposal. Disposal of EB into the sanitary sewer (sink drains) is prohibited. Working solutions can be be rendered non-toxic as the last step of your experimental process. If you choose to detoxify EB in your laboratory, campus policy requires that you meet all the following conditions: