-Advertisement-
  About AE   About NHM   Contact Us   Terms of Use   Copyright Info   Privacy Policy   Advertising Policies   Site Map
   
Custom Search of AE Site
spacer spacer

Agarose Gel DNA Quantitation

By J. Kirk Brown



Type of Entry:

  • Lesson/class activity

Type of Activity:

  • Hands on Activity
  • authentic assessment
  • community outreach
  • review and reinforcement of concepts
  • group/cooperative activity

Target Audience:

  • Biology
  • Advanced Placement/International Baccalaureate, Advanced Biology
  • Anatomy/Physiology
  • Integrated Science 1,2,3,4
  • Genetics, Biotechnology

Notes to Teacher:

This activity is not designed to be the students first exposure to electrophoresis. Students should have previously analyzed digested DNA and determined the size of the fragments by semilog graphing. This activity can be used by a biology class having just completed electrophoresis or it can be part of a culminating activity in a senior level advanced biology or biotechnology class. How this activity is utilized will depend upon the level of the students and the focus of the experimentation. The activity can be completed using photographs or photocopied pictures of gels. The use of pictures enables students to practice gel quantitation without having to run the gels. This technique can also be taught as part of a research project that utilizes the skill to help answer the research question. Note: Adhere to appropriate safety guidelines when using ethidium bromide.

Required of students:

Students will be looking at agarose gels, or photographs of agarose gels, to compare a known DNA sample (Lambda/HindIII) to an unknown sample to determine the quantity and size of an unknown DNA sample. Students at the advanced level might be using this technique as part of a larger assignment.

Preparation time needed:

If teachers are going to make a class set of pictures, it will take approximately one hour to take a class set of pictures after the gel has run. To prepare the known the teacher should prepare a Lambda HindIII digest (see student instructions) or purchase one already digested and quantified( I recommend this highly). Load 10 microliters of 0.1 ug/ul Lambda HindIII plus one microliter of loading dye into lane one of the gel. This will load 1 ug of total DNA into the well. Use any other DNA/restriction enzyme combinations to produce some unknown(to students) fragments. These can be run in the other lanes. Run at 70 volts for approximately 2 hours in 0.8% agarose. Stain with ethidium bromide and photograph. If this is being done in Advanced Biology it would be appropriate to have students do all of the work themselves. (See list of DNA and enzymes at end)

Class time needed:

Class time varies from one hour if photographs are used, to three hours if gels are run and students stain and photograph their own gels.


Background

What question does this activity answer for students?

Gel Quantitation is a technique used by research scientists to get an estimation of the size and quantity of DNA fragments in an agarose gel. The technique involves the comparison of a known quantity of DNA to an unknown quantity of DNA on an agarose gel. The gel is stained with ethidium bromide and photographed. The brightness of a particular fragment, as well as the distance traveled, can be used to estimate the quantity and size of a particular fragment as compared to a known sample such as Lambda cut with HindIII.


Project

Gel quantitation is a simple technique used by research scientists to quickly estimate the quantity and size of DNA fragments in a particular sample run on an agarose gel stained with ethidum bromide. If methylene blue is used it will work in a similar fashion. A known sample is run in lane one. This sample can be any known sample but I prefer to use Lambda/HindIII. 10 microliters of 0.1 ug/ul are loaded into lane one. Since each fragment length is known, it is possible to estimate the percentage of the total Lambda DNA. The brightness of the bands is compared and students can estimate the quantity of DNA in the unknown bands by comparing with the known fragments from the Lambda/HindIII lane.

Lesson/Activity

Materials:

  • Electrophoresis box
  • Power supply
  • Gel tray and comb
  • Lambda DNA at known concentration
  • Restriction enzyme HindIII
  • Other Restriction enzymes
  • Other sources of DNA
  • micropipet
  • loading dye
  • agarose
  • 1X TBE buffer
  • ethidium bromide
  • transilluminator/camera
  • 1.5 ml eppendorf tubes

Activity

Procedures:

  1. Have each group of students pour a 0.8% agarose gel. This can be done by adding 0.8 grams of agarose to 99.2 ml of 1X TBE buffer and microwaving on medium low for 2-3 minutes or until clear( make sure not to seal the container while microwaving).

  2. Have each group of two students (if you have enough gel boxes etc...) use a source of DNA and cut with restriction enzymes( Lambda BamH1, Lambda EcoR1, pBR322 Hind III are just a few possibilities). Set up each digestion by adding 2 ul of source DNA at .5 ug/ul and 1 ul of 10X restriction buffer, 1 ul of restriction enzyme and 6 ul of double distilled water. Incubate overnight at room temperature or 2 hours at 38 C.

  3. After the restriction enzymes have cut the DNA, load 1 ul of loading dye into each sample. Adjust the micropipet to 11 ul and load the samples in lanes 2-8. In lane one add the Lambda/HindIII digestion (10 ul of 0.1 ug/ul sample for a total of 1 ug of DNA) plus one ul of loading dye( 11 ul).

  4. Run at 70 volts for approximately 2 hours for best results, or 100 volts for one hour if time is an issue.

  5. Stain the gels for approximately 5 minutes in ethidium bromide and de-stain in water for 2 minutes.

  6. Photograph gels

Analysis of Pictures

  1. Look at the Lambda/HindIII lane

  2. Since each band size is known, some estimations can take place. The slowest band is the 23,130 bp band while the fastest is usually not visible at 125 bp in size.

    23,130
    9416
    6557
    4361
    2322
    2027
    564- usually not visible
    125- usually not visible

  3. Since a known quantity of the Lambda HindIII was loaded into the lane it is possible to estimate the quantity in just one fragment. By knowing the total length of Lambda a percentage can be determined.

    23,130/ 48502        = 48%          564/ 48502          = 1%
    9416/ 48502          = 19%          125/ 48502          = .2%
    6557/ 48502          = 13%
    4361/ 48502          = 9%
    2322/ 48502          = 5%
    2027/ 48502          = 4%
    

  4. Since 1 microgram of DNA was loaded into the lane to begin with, it can now be used to determine the quantity of the Lambda/HindIII.

    Example: 23130 is the slowest band and it is also the brightest. Since 48% of the total DNA is in band 23,130 and there was 1 ug of total DNA, therefore 48% of 1 ug =.48 ug of DNA in band 23,130.

  5. Students should then estimate the rest of the known bands. They then can compare the brightness of the known bands to the unknown bands. If the brightness of a known band is close to the brightness of an unknown then an estimation of the total number of micrograms can be made.


Method of Assessment/Evaluation

Each student should label their photograph with the size estimation and quantity of a given band of DNA. If this is a lower level class then students can practice on a number of different pictures and give estimations of the quantity of DNA in each of the unknown samples. Another possibility is to have each group research how many restriction sites a particular plasmid has and also determine the quantity of each fragment. They can present this data to the class as a research symposium. A third possibility is to have the students use this technique to estimate the size and quantity of a piece of human DNA released from its bacterial vector DNA. This focus is one that works very well because it is in context and not just an exercise on gel quantitation. By utilizing the research approach, gel quantitation becomes a skill necessary to carry out the research at hand. All levels of students should also prepare a laboratory report that gives all findings and data.


Extension/Reinforcement/Additional Ideas

As previously mentioned, this activity can be used at various levels. In most high schools it will probably be best suited as an advanced biology lab. In schools that use electrophoresis in the freshman and sophomore years it is possible to use it then. This can be conducted as a dry lab with photographs, or it can be a hands-on lab with students running gels and doing the work themselves. There are some good extension activities that can be conducted using this lab as one in a series of activities. An activity that I find rewarding and fun is the involvement of the students in real research. I obtained a human cDNA clone from Research Genetics (http://www.resgen.com). These can be purchased online. The students used the WWW to research information and order the clone. They contacted a research scientist at Lawrence Livermore National Laboratory and received help in designing an experiment to determine the sequence for the given clone. They used gel quantitation to roughly determine the human insert size and quantity of the insert. They then proceeded to do restriction analysis on the insert to see if the information that they obtained was possibly correct given the proper fragments. Students then used the internet to upload the data gathered. There are many more variations and options that utilize this simple technique. For a more accurate quantitation, a spectrophotometer is used to determine exact amounts. Catalog sources of DNA and Enzymes: Edvotek Wards Biological Carolina Biological


Fellows Collection Index


1996 AE Collection Index


Activities Exchange Index


 
Custom Search on the AE Site

 

-Advertisement-