DNA question Bluescript

Question

We loaded purified plasmid DNA into 4 individual lanes of an agarose gel. Using the diagram on the right, draw bands of appropriate size where you would expect to see them after electrophoresis. The size of theDNA ladder bands is given on the left. Recall the possible virus insert sizes are: 125, 564, 2027, 2322, 4361 6557, 9416 and 23130 bp. Also recall that the plasmid is 3kb (3000 bp).

 

Lanes are divided:

 

pBluescript II / pBluescript II with 125 bp insert/ pBluescript II with 2027 bp insert/ pBluescript II with *bp insert

 

I cannot seem to attach the table, however have written the

Answer 1

DNA ladder markers that are commonly used are 123 bp ladder and 1 kb ladder. I do not know which one you are using. 

 

pBluescript is about 3 kb and therefore plasmids with insert size 125, 564, 2027, 2322, will be as follows:

 

3.1 kb,

3.5 kb

5 kb 

5.3 kb.

 

5.3 kb will run higher than rest, 5kb next, 3.5 kb much lower than these two bands and 3.1 kb even lower.

 

I am assuming that you have linearized the plasmid with single cut restriction enzyme, if not then they would run differently. Uncut circular plasmid DNA runs differently then linear plasmid DNA.

 

If you have inserts of 4361 6557 respectively then band size would be 7.3 and 9.5 kb, they would run on very top of the gel. 

 

9416 bp insert would give a band of almost 12 kb and it would barely enter the gel.

 

I do not think that pBluescript can take 9416 insert easily and definitely not 23.1 kb.

 

Band separation on agarose gel also depends on the percentage of agarose gel and the the speed of running. For bands higher than 3 kb, 0.7% agarose gel run very slowly overnight would give relatively accurate size. 

 

One should run a linearized  plasmid without insert as control for accuracy of insert.

 

Hope this is helpful and clear, post/ask again if you have further questions.

 

Best,