For this question assume that you have no other information (i.e., you didn\'t t

Question

For this question assume that you have no other information (i.e., you didn't test to see how well the probe was labeled, etc.) Lets suppose that the Southern blot was completely blank. No labelled band was observed. Give two possible reasons how/why this could have happened. Explain for each of your answers, how you could test to see if that is what occurred.

Let's suppose that for the Southern blot, several bands were observed. You were expecting a single band. Give two reasons how/why this could have happened.

Explanation / Answer

1) Southern blot involves the technique in which fragments of DNA to be identified are transferred to another medium, where hybridization may be carried out. So it is possible that there was failure in the transfer of DNA to the medium. To detect this, one can use the method of probe hybridization.

--> Southern blot also involves denaturation of DNA for hybridization to take place. So it is possible that the DNA did not denature enough or at all. To detect this, one can use X Ray Crystallography, and compare the paritally denatured DNA with a reference structure.

2) In case of several bands being observed, one of the reasons for such to happen is when the probe works in a way one does not expect. In case where we want a single band to occur, we know this can happen when the probe binds with a single strand of DNA. However, if multiple bands are observed this is likely because the probe hybridizes to several sequences which are similar to the desired DNA strand.

Another reason could be conducting the experiment in not so ideal conditions, i.e. improper temperatures used, not enough or too much baking etc.

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