Question

Imagine you must run a run on a genomic DNA extraction. Start by preparing the agarose gel and forget to add ethidium bromide to the solution. What consequences will you face when analyzing the results?

Imagine that you must perform a transformation using thermal shock. But when doing the preparation, you realize that you do not have calcium available for the procedure. Can the transformation be carried out without the calcium? What will be the consequences?

Answer 1

1) EtBr (Ethidium Bromide) is a common dye used in the genomic DNA extraction protocols. It is an intercalating agent, which binds to the spaces between double stranded DNA base pairs, and thus it can alter the overall charge, weight, construction and conformation of the DNA. It is commonly used in agarose gel electrophoresis, which is for the separation of DNA fragments.

How EtBr works?

1) It binds between basepairs in the DNA and thus increases the weight and friction of that fragment of DNA. Thus if the fragment has more basepairs, it has more binding of EtBr and thus have more friction. Thus friction is directly proportional to the size of DNA and the relation is described by reptation theory. So fragments having more friction moves slowly and those heavy bands are seen in the top of the gel with less mobility.

2) EtBr has the main property of fluorescence in the presence of UV. Thus the DNA fragments where EtBr are attached, ran through the gel can be seen under UV light. It can either add to the gel or to the loading buffer also.

EtBr is mutagenic as it can alter the DNA conformation.

If EtBr is not added to the gel, the movement of fragments may increase and it can overrun in the specific time as compared to the added scenario. And the bands will not be visible under UV as dye is absent.

But don’t worry.. we can also add EtBr to the loading buffer also.

  

2)Transformation is done to incorporate a foreign DNA into a host cell which is commonly a bacteria. Heat shock method is the most common type used. Transformation become possible only if the host become competable to a foreign DNA. A competence cell is prepared by treating the cells with a solution containing high amount of calcium. Why calcium is important?

1) It make pores in the bacterial surface and thus a cell become more permeable.

2) It depolarizes the cell surface and thus the negative potential inside the cell decreases and thus a negatively charged DNA can easily enter inside the cell.

The cell becomes competent only if treated with calcium, then incubating cell and foreign DNA in ice, and then giving heat shock.

If calcium is not added, then cell cannot be perforated and thus transformation wont occur.