Question

1. Imagine you have a plasmid and you cut it with three different restriction enzymes (enzymes 1, 2, 3). You then run these fragments through a gel and get this separation.
   1. Label the positive and negative terminals on this gel with a (+) and (-) sign.

2. If you were working with a single plasmid, how many cut sites did restriction enzyme 1 have? Explain?

3. When creating a gel, we stain it with Ethidium Bromide or Gel Red. What is the purpose of this? Explain.

2- When separating plasmids from proteins and ribosomes, why do you add N Buffer and THEN centrifuge? Why not just centrifuge? What is the advantage of adding the N Buffer?

1. I give you 100mL of a 3 M aqueous glucose stock solution. I need you to dilute it to make 100mL of a solution that is 0.03M. To accomplish this task, you have 2 additional 100 mL graduated cylinders and however much distilled water you need. The graduated cylinders are not really accurate at measuring any volume less than 5mL. (10 pts)

1. How would you dilute 3M stock solution into 0.03 M solution? Explain strategy.

Show your calculations:  

Answer 1

Answer: The labelled agarose gel electrophoresis is shown below.

If there is a single plasmid, the number of cut sites the restriction enzyme has is one. The number of the cut site for a restriction enzyme depends on the specific sequence present in the DNA molecules. Many plasmids are smaller circular DNA molecules and hence can have one cut site for a specific restriction enzyme.

Ethidium bromide is an intercalating agent that inserts between the base pairs in the double helix. Ethidium bromide absorbs UV light and emits at 590 nm. This is used to tag the DNA molecule.

Buffer resist the slight changes in the pH level. When the plasmids are separated from proteins and ribosome, a buffer of pH near to the physiological pH (about 7.0) is used. Extreme pH can disrupt the biomolecules leading to unsuccessful separation. N buffer allows proper separation of plasmid, proteins and ribosomes. This makes the separation of DNA, proteins and ribosomal particles easy through differential centrifugation. As per the structure and molecular mass different molecules are easily separated in centrifugation using N buffer.    

To prepare 100 ml of 0.03 M of glucose solution from 100 ml 3 M stock solution,

1. Take 5 ml of 3 M stock solution and make volume up to 50 ml using distilled water in a separate graduated cylinder. This makes 50 ml of 0.3 M glucose solution.

2. From the 0.3 M glucose solution, take 10 ml and make the volume up to 100 ml in a separate graduated cylinder. This makes 100 ml of 0.03 M glucose solution.

The 3 M stock solution can be diluted to prepare a 0.03 M solution using the following calculation:

100 ml of 3M glucose solution

1 ml of 3 M glucose solution when diluted with 99 ml distilled water or a 100 times dilution, the resultant solution is 100 ml 0.03 M glucose solution.