Question

questions for Biology lab

An agarose gel electrophoresis assay performed with high quality will NOT have a DNA ladder with which of the following characteristics

Bands that are stained well (easily visible under UV light)

Bands that are separated enough

Bands that are blurred

Crisp and rectangular bands

During which step of a PCR reaction do the dNTP molecules get incorporated into a DNA strand

Extension

Annealing

Denaturation

None of the above

Which of the following steps does not get cycled/repeated in a PCR reaction?

Annealing

Predenaturation

Denaturation

Extension

A factor to consider when selecting the optimal MgCl2 concentration to use in a PCR reaction is which of the following

Yield

Specificity

Robustness

All of the above

A student needs to prepare a 25ul PCR reaction that reverse primer at a final concentration of .4uM. The student works in a lab that keeps the stock concentration of reverse primer at 21uM.

.33ul

3.3ul

4.7ul

.47ul

None of the above

A student has pipetted 2ul of a 50mM solution of Magnesium Chloride into a container. After pipetting this volume, the container had a final volume of 19ml. What is the approximate final concentration of Magnesium Chloride?

.005M

.05mM

.76mM

.00076mM

None of the above

Higher annealing temperatures in a PCR help reduce what?

Nonspecific products

Template denaturation

Template renaturation (DNA Coming together to form double stranded DNA

None of the above

Answer 1

Ans 1) Bands that are blurred

DNA ladder are basically markers which are used in the agarose gel electrophoresis and helps in identification of approximate molecule size. This helps in making a comparison between the DNA that is run on the gel while comparing the ladder. It is observed under UV light and are separated properly to make analysis. The ladder bands are quite crisp and rectangular in shape but if the assay is not done properly, the DNA ladder bands might appear blur.

Ans 2) Extension

This is one of the major and most important cycle that occurs in the process of PCR. During this process the temperature is raised to 72 degrees C and is the step where the dNTP binds to the elongating chain an d helps in DNA elongation.

Ans 3) Predenaturation

The stages after pre-denaturation include the process of denaturation, annealing and elongation which are three phases that are repeated in a cycle. The pre-denaturation is also known as the stage of initialization and is required only once for heat activation. The initialization step includes holding the thermostable polymerase for 1 to 10 minutes at 94 - 96°C.

Ans 4) Specificity

The MgCl2 is basically used as co-factor and helps in catalyzing the PCR. With higher concentration of the MgCl2, the productivity of the Taq polymerase increase but with productivity the specificity will be less leading to production of smears. Hence the concentration must be optimized in a way that specificity is not hindered and productivity is also maintained.