Why is it necessary to boil the probe before you use it in your southern blot? (

Question

Why is it necessary to boil the probe before you use it in your southern blot? (b) (3 points) How did you make the DNA strands on the gel single stranded? (c) (3 points) What is the function of the blocking reagent? (d) (3 points) What is the function of the neutralization solution?

For the hybridization step in the Southern blotting, below is the sequence of the probe that was used. The Tm is given for this probe assuming a perfect match for the target sequence.

ACTTACTgCTggCTAATCgggCTATTC    Tm=65OC

By mistake, I perform the hybridization at 85OC instead of 58OC. Explain how this would affect my results. (b) By mistake, I perform the hybridization at 45OC. Explain how this would affect my results. (c) Explain what is meant by the term stringency. (d) How can the stringency be adjusted?

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.) (a) (10 points) Let’s 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. (b) (6 points) 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

a. Probe is boiled to denature it.

b. Alkaline agarose gel can be used to  make the DNA strands on the gel single stranded.

c. Blocking is required to prevent nonspecific binding of the detection antibodies during subsequent steps.

d.  neutralizing solution is added causing the denatured genomic DNA to precipitate, while the circular, covalently closed plasmid DNA reanneals.

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