The Effect of Temperature on Restriction Enzymes

Purpose

The purpose of this study is to investigate the effects of temperature on the action of a restriction enzyme.

 

Hypothesis

DNA fragments cut with a restriction enzyme maintained at 60 degrees C will produce the smallest fragments as indicated by electrophoresis.

 

Background

Enzymes are special proteins that affect the rate of chemical reactions. Enzymes are made by saliva, the stomach, the pancreas, and the small intestines. In the process of digestion the enzymes become very active. Enzymes help split larger molecules into smaller ones.

In 1822, William Beaumont, a U.S. army surgeon, had a very unique chance to study the digestive system because a soldier was shot in the stomach. With the cooperation of the patient, Dr. Beaumont watched the enzymes do their work. He found out that enzymes were a protein and the in lining of the stomach is made up of chemical resistant tissue.

. Without enzymes food would digest very slowly. Temperature can change the structure of the enzymes so that they do not function.

Enzymes can increase the rate of digestion up to a trillion times faster than the same reaction without enzymes. Enzymes reaction time increases when thermal energy is applied until it reaches a certain point and then changes the structure of the enzyme. There are many factors that can affect the enzyme reaction rate. Variations in the concentration of substrate molecules, temperature, and pH, just name a few, are all factors that affect enzymes.

 

Materials



Enzyme and DNA

Distilled water

Boiling water bath or microwave oven

Graduated cylinder

Beakers

10 micropipettors

6 Gel Electrophoreses Chambers

Buffer

Power Source

Funnel


Procedure

I. Making the agrose gel:


Mix agrose powder using 176.4 m. of distilled water and 1.44 gm of
powder and 3.6 ml of buffer solution to make 180 m. of mixture.

Let it sit for 5-15 minutes or until gel-like.

II. Temperature of enzymes:

Add DNA and enzymes using micropipette

Control the following temperature with water baths.

Sample A - 0 degrees C.

sample B - 37 degrees C.


Sample C - 60 degrees C.

Sample D - 100 degrees C.

IV. Setting up electrophoreses apparatus:

Attach lid to the electrophoreses tray (one negative one positive) and
plug in clips to the power source.

Make wells and trenches with combs.

V. Experiment:

Transfer DNA/enzymes mixture into the wells.

Set power source to 50 volts and turn on.

After 2 hour turn off power supply.

Repeat for each temperature. Repeat entire experiment 3 times.

VI. Finding results:

Measure the distance the DNA travels through the gel.

Calculate a mean for each group and graph.





Results

Mean Electrophoresis Distance vs. Temperature

(C) Vs (cm)

0.0 C Vs 1.6 cm

37.0 C Vs 0.06 cm

60.0 C Vs 2.4 cm

100.0 C Vs 0.0 cm

Conclusion

The data did support the hypothesis. Group C (60 degrees C.) did have the longest lines in the gel.

 

 

Links


Carolina Research Supply Company


Electrophoresis Total Lab