Electrophoresis of Nucleic Acids

An experiment on nucleic acid is conducted to characterize and quantify them. several quantities of reagents are used in the molecular biology experiment to see if the DNA molecules of interest will move within the course of the series of procedures. The technique which will be used is known as electrophoresis, which is useful in determining the size of DNA and RNA molecules, assess the purity of isolated DNA and RNA and estimate the concentrations of the DNA and RNA samples. The experiment will be based on the fact that log10 of the base pair length of DNA fragments has a linear relationship with the distance that the fragments move through an agarose gel.

Materials and methods

Method 1

A micropipettor and a small microcentifuge tubes, numbered from 1 up to 40 will be used. each of the microcentrifuge tubes will have two dyes of different charge and molecule weight. The micropipettor will be used to transfer 10 µL of the dye into a well in a practice agarose gels. The wells of the agarose gel will be connected to a power supply and after 45 minutes, the color emitted by each one of dyes will be investigated to determine their identity. The results will then be tabulated.

A ruler will then be used to measure the distance between the center of the well and the center of the dye band for each of the dyes. The distance of the amido-black will also be measured. The results will also be tabulated together wit the colors of the separated dyes in the second row of Table.

Method 2

Plasmid DNA that has been cut with a restriction enzyme HindIII will be put in 26 microcentrifuge tubes labelled A through Z.

A micropipettor will be used to add three spots on a 1 inch strip of Parafilm

o spot #1 will be 5 µL of water and 1 µL of sample buffer.

o Spot #2 will be 1 µL of 0.5 µg/µL λ/HindIII DNA, 4 L water and 1 µL of sample buffer

o Spot #3 will be 5 µL of the unknown DNA and 1 µL of sample buffer.

Results

The table below displays the Electrophoretic mobility of dyes in an agarose gel

First Unknown Dye

Second unknown Dye

Amido - Black

Distance (cm)

1.8 cm

1.5 cm

1.8 cm

Color Dark Blue

Blue

Green

R1

Value

1

- 0.83

1.00

The table 11.3 displayed above shows the Electrophoresis of standard and Unknown DNA fragment in a 1.5% agarose gel

λ DNA Fragment size (bp)

Log10

λ DNA Fragment size (y)

Distance λ DNA Fragment Moved (cm) (x)

Distance Unknown DNA Fragment moved (cm)

23,130

4.36

0.4

9,416

3.97

2.2

6,557

3.82

2.5

4,361

3.64

2.9

2,322

3.37

3.3

2,027

3.31

4.2

564

2.75

4.4

125

2.10

6.3

Equation:

A scatter plot of Log10

λ DNA Fragment size as the dependent variable (y) and Distance λ DNA Fragment Moved (cm) as the independent variable is displayed blow. The line of best fit of the form y = mx + b and the R2 are displayed below

The equation of line of best fit is

Whereas the R2

value displayed is

Interpretation

Because the gradient is negative, the two variables are inversely related. Based on the equation, a unit change of in the Distance λ DNA Fragment move causes a 0.398 unit change if the Fragment size, in the opposite direction.

As for the R2, it means that the independent variable, Distance λ DNA Fragment Moved determines 94.4%of the dependent variable – Log10 λ DNA Fragment size.

Conclusion

The two dyes which were separated were Brilliant Cresyl Blue, because it is of color Dark Blue and an R­1

value of -0.85 (in the experiment, the value was -0.83).

The conclusion of the charges and molecular weights of the dyes in the unknown are inversely related. In other words, heavy dyes will move shorter distances and lighter ones will move linger distances.

If the gel and buffer used were run at pH 11 instead of 7, then it means that it will be more basic and no longer be neutral, and therefore, the results will not be reliable.

The correlation between the log10 values for the size and the distance that they travelled in the 1.5% agarose gel was very high, with a correlation coefficient of  

 = - 0.972

The negative sign is because they are inversely related.

Using the linear equation above, we can calculate the length of the unknown DNA fragment

The size is 2.80784cm at 95% confidence level.

When 1.0% agarose was used in the experiments, the correlation between the distance travelled and the weight would be less than what was calculated.