Electrophoresis and Gel Quantitation

Advanced Preparation

• Time: One day before lab; set up digest(15 minutes). Run gel; 1 hour
• The production of Pictures for students to practice on: Pictures can be produced by using various restriction enzymes on Lambda DNA or any other small source of DNA. If you use a large source you will get too many fragments and not much separation between fragments. One suggestion is to use Lambda cut with BamH1, Lambda cut with EcoR1, Lambda cut with Hind III. These enzymes are cheap and easy to obtain. Lambda is also inexpensive. If you have other sources of DNA and enzymes it will work just as well as long as the concentration is known. To produce the Lambda at a concentration of 0.1 microgram per microliter it is necessary to add 10 microliter (µl) of DNA at 1microgram (µg) /µl, 10µl of 10x restriction buffer, 2µl of HindIII restriction enzyme at 1unit/µl concentration, and 78µl of double distilled water (sterile). Incubate overnight at room temperature. The next day place into a frost free freezer and freeze until ready to use or use before DNA is frozen.
• Follow the same procedure for the other DNA and enzymes by just substituting the other enzymes. This will produce enough for 10 samples each.
• To run gels (0.8 % gel) add 10 µl of Lambda HindIII digest and one µl of Loading dye. Draw up all eleven µl of mixture and place into lane one.
• Repeat the procedures for the other digests and add them to the next two lanes.
• Add another Lambda HindIII to the last lane.
• Run gels at 74 volts for one hour.
• Stain with Ethidium Bromide for 5 minutes and rinse for 1 minute in water.
• Photograph gels

Introduction

This laboratory will enable students to develop an important laboratory skill. Gel quantitation is important for scientists working with DNA on a daily basis. If a restriction digest was carried out on a cosmid or other large piece of DNA and a particular sized fragment was desired, not only the size would be important, but the quantity of DNA in the fragment would also be important to estimate. A technician would know if they needed to cut more DNA in order to account for the loss of DNA in the gel purification protocol. This technique provides an estimation of the amount of DNA in a particular fragment. A more accurate method is to quantitate with a spectrophotometer over a range of wavelengths.

Student Objectives

• The student will correctly estimate the amount of DNA in a unknown band of DNA on a gel.
• The student will develop an experiment that would utilize gel quantitation as part of the laboratory.

Class time needed

One class period to complete initial estimations, one class period to do extensions.

Materials

1. Pictures for students to use: at least one per student
2. Rulers
3. Calculators

Recipes for Making gels

0.8% agar is made by adding 0.8 grams of agarose and 99.2 grams of 1X TBE buffer. Microwave on medium for l-2 minutes. DO NOT CLOSE. PUT PAPER TOWEL OVER TOP OR INTO TOP OF CONTAINER.

Procedures

• Having many other different samples for students to practice on is very helpful. If there are any laboratories near your school you could request some pictures to use in your class.
• Have students calculate the amount of all of the bands on a gel.
• This laboratory is nice to follow up with gel electrophoresis and graphing using semilog paper to calculate the size of DNA fragments.