This is apart of an rfp gene lab done in an AP Biology course. (Using gel electr

Question

This is apart of an rfp gene lab done in an AP Biology course. (Using gel electrophoresis with a marker, R+ tube (has restriction enzymes) and R- tube (no restriction enzymes).
Q1) why might the enzymes work best at 37C? Why should the enzymes then be placed in the freezer?
Q2) The DNA is not visible as it moves through the gel. The loading due contains the three dyes initially separated. Why is it useful to use the loading dye in this lab?
Q3) in the picture:
seconds to ntrifuge so that Figure 4A.4: Leads from electrophoresis in power supply located near the face of the gel. nts of buffer tely under the I allow the he wells in the follows: 10. Turn on the power supply and set the voltage to 130-135v 11. After two or three minutes, check to see if the purple loa After (bromophenol blue) is moving toward the positive (red) moving in the other direction-toward the negative (bla the electrical leads to see whether they are plugged in to correctly NA ladder (M), each sample, the If needed, ove the well. unusable. avoid getting will sink into STOP AND THINK: NA ladder serves as a standard because it contains a mt of DNA molecules of known sizes. By running your samples and DNA ladder side by side in your gel, you can estimate the actua in base pairs of unknown molecules. The DNA Ladder Diagram 4A) shows 10 DNA bands of known sizes. Using this informaton can you predict the positions of DNA bands produced by the possible products found in the R-and R+ tubes by indicatinge position on the DNA Ladder Diagram? ver the ads to the hode (+) to DNA samples and the DNA ladder are not visible on the How might the DNA be made visible once the gel el complete? lectro 12. Your teacher uill

Explanation / Answer

Ans. #1. Almost all the biochemical system has evolved through millions of years to functional optimally at specified pH, temperature and other physiological conditions of the host organism, in this case 37.00C – the human body temperature.

In terms of molecular biology, all the amino acids and their functional groups in the enzyme are orientated in proper 3D conformation required for the catalytic activity of the enzyme. The “catalytically active form” of enzyme is stable only at narrow range of temperature- optimally at 37.00C. Therefore, enzymes work best at 37.00C.

# Change in temperature (increase or decrease) changes the kinetic energy content of the constituent atoms and molecules in enzyme. It further interferes with non-covalent interactions including van der Waals interactions, hydrogen bonding pattern, ionic interactions, etc. that would disrupt the normal 3D conformation of enzyme and in turn, the “catalytically active form” of enzyme. Therefore, deviation from optimum temperature usually leads to lowering and/or loss of enzyme activity.

# Enzymes are stored at low temperature, preferable at or below -20.00C. At such low temperature, the enzyme becomes inactive but retains the ability to become active when thawed to normal room temperature. Therefore, enzymes are stored in freezer to preserve its catalytic activity for prolonged time.

#2. The purpose of using loading dye-

I. The loading dye binds to DNA molecules and increase its apparent density. So, the DNA molecules remain settled in the gel and move within the gel. If loading dye is not use, the DNA molecules may come out from gel into the buffer due to buoyancy. In that case, no DNA bands specific to each well would be observed.

II. DNA solutions are colorless. The addition of loading dye (blue-colored) provides the mean of tracking the movement of DNA molecules through the gel.

#3. Picture- Stop & Think:

A. DNA ladder, R- and R+ tubes: The exact relative masses of bands from ladder DNA is not mentioned. Moreover, the exact information on size of DNA molecules being digested, number and type of restriction sites on DNA, restriction enzyme is not mentioned. So, it won’t be possible be predict the exact location of R- and R+ DNA bands in the gel.

However, the bands from R- tube would be close to the wells because of its greater mass. It produces a single band because the DNA molecule remains intact, but not cleaved into fragments.

The R+ tube may produce two or more bands because restriction cleaves the DNA into two or more fragments of smaller size. SO, the resultant bands would be located away from the well depending on relative masses of DNA fragments.

#B. DNA is made visible by the addition of EtBr to the gel and viewing it under UV transilluminator.

EtBr chelates between the DNA bases. When exposed to UV light, the resultant DNA-EtBr complex appear golden yellow color- thus revealing the location DNA or bands in the gel.    

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