Someone help me pleaseeeeeeee

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discovery
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Someone help me pleaseeeeeeee

 
Figure Q10-14 shows the recognition sequences and sites of cleavage for the restriction enzymes Sal I, Xho I, Pst I, and Sma I and a plasmid with the sites of cleavage for these enzymes marked.
 
 

 
 
Figure Q10-14
 
A.        After which of the following treatments described in choices 1 through 5 can the plasmid shown in Figure Q10-14 be recircularized simply by treating with DNA ligase? Assume that after treatment any small pieces of DNA are removed, and it is the larger portion of plasmid only that you are trying to recircularize.
 
            After digestion with
            1.         Sal I alone.
            2.         Sal I and Xho I.
            3.         Sal I and Pst I.
            4.         Sal I and Sma I.
            5.         Sma I and Pst I.
 
B.        In which of the cases 1–5 can the plasmid be recircularized by adding DNA ligase after the cut DNA has been treated with DNA polymerase in a mixture containing the four deoxynucleotides? Again assume that you are trying to recircularize the larger portion of plasmid.
 

The FFM
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A.        After which of the following treatments described in choices 1 through 5 can the plasmid shown in Figure Q10-14 be recircularized simply by treating with DNA ligase? Assume that after treatment any small pieces of DNA are removed, and it is the larger portion of plasmid only that you are trying to recircularize.
 
            After digestion with
            1.         Sal I alone. -  recircularized yes - 
            2.         Sal I and Xho I.  recircularized becuase Sal I and Xho I have compatible ends - see http://www.neb.com/nebecomm/EnzymeFinderSearchByEnd.asp?txtSequence=TCGA&selDirection=2&radSearchType=0
            3.         Sal I and Pst I. - no not compatible sticky ends
            4.         Sal I and Sma I. - no you cant ligate blunt and sticky ends without first blunting the sticky end
            5.         Sma I and Pst I. - no same reason as 4
 
B.        In which of the cases 1–5 can the plasmid be recircularized by adding DNA ligase after the cut DNA has been treated with DNA polymerase in a mixture containing the four deoxynucleotides? Again assume that you are trying to recircularize the larger portion of plasmid.
 
all of them as if they are not already blunted the polymerase and nucleotide mix will fill in the sticky end overhangs (if any) and allow you to ligate all the blunt ends that result
 
 
 

discovery
discovery's picture
Well thank you for the answer

Well thank you for the answer, it was simple and i got a clue as well answer for the same. Your help is highly appreciated.

Jason King
Jason King's picture
 

 
            After digestion with
            1.         Sal I alone.
            2.         Sal I and Xho I.
            3.         Sal I and Pst I.
            4.         Sal I and Sma I.
            5.         Sma I and Pst I.
 
B.        In which of the cases 1–5 can the plasmid be recircularized by adding DNA ligase after the cut DNA has been treated with DNA polymerase in a mixture containing the four deoxynucleotides? Again assume that you are trying to recircularize the larger portion of plasmid.
 

 
Answer: Certainly 1 and 2. And if you were trying to do the two digestions at the same time, then I guess 4 and 5 as well, since SmaI is not compatible buffer-wise with either Sal I or Pst I .  ; )

The FFM
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parvoman wrote:

parvoman wrote:

 
            After digestion with
            1.         Sal I alone.
            2.         Sal I and Xho I.
            3.         Sal I and Pst I.
            4.         Sal I and Sma I.
            5.         Sma I and Pst I.
 
B.        In which of the cases 1–5 can the plasmid be recircularized by adding DNA ligase after the cut DNA has been treated with DNA polymerase in a mixture containing the four deoxynucleotides? Again assume that you are trying to recircularize the larger portion of plasmid.
 

 
Answer: Certainly 1 and 2. And if you were trying to do the two digestions at the same time, then I guess 4 and 5 as well, since SmaI is not compatible buffer-wise with either Sal I or Pst I .  ; )

This is obviously an exam question and they dont mention double or sequential digests or buffer compatibility so I just advised discovery according to the info given which as I read it assumed all the enzyme cut.

Shampa
Shampa's picture
Whatever I had in mind,

Whatever I had in mind, Parvoman has answered more than that. you can follow that clue.

varsha
varsha's picture
Hi discovery.

Hi discovery.
This sounds like a test paper.  We could help you understand the question or the concept behind the question. I am not sure we are helping you out by doing the homework/test for you. 
Varsha