Generating a sequence for a Digested Plasmid

[LyScience]

Hi..

 

I'm just wondering if anyone could help me with how would you generate a sequence for a plasmid that has been digested using Xhol/EcoRI

 

Thanks in advance

[zule]

Do you want the sequence of the primers for amplifying the fragments of the plasmid by PCR or what exactly do you want?

Is this homework? Because such a specific question is not usually something that you “wonder” while you are having a beer

[LyScience]

Well, maybe I choose the wrong verb, but it's not really a homework, it's a question form a practice exam and I want to make sure that I'm able to do it before the exam. And yes i want to know how to generate the sequence of the primers.

 

Thanks:rolleyes:

[zule]

Xhol/EcoRI is a restriction enzyme which recognizes the DNA sequence 5’… CTCGAG…3’ and cuts this sequence between the T and the C.

 

Taking into account that the DNA strand is double, the plasmid will become lineal, doble-stranded, with single strand ends: 3'...AGCT...5' and 5'...TCGA ...3'

 

So, we can use the primer TCGA and AGTC to amplify starting with those free ends.

 

It would be much more easily understable with drawings.

Edited October 25, 2009 by zule

[CharonY]

Actually I do not think that is what is meant. Beside the fact that such short primers are usually not very useful and that the ends will be different. Just from memory XhoI recognizes CTCGAG but generates sticky ends, i.e. 5'C TCGAG3' and 3' GAGCT C 5' respectively (as mentioned above). However, EcoR1 recognizes GAATTC and cuts between G and A (again sticky ends): 5'G AATTC3' and 3'CTAA G5'.

The space indicate cuts. What you normally do is that you know the sequence beforehand, then you digest the DNA virtually using the above information and see what fragments are expected. In addition it yields a little bit of sequence information, but this is most of the time obsolete.

 

If the fragments in question are cloned into a vector for sequencing purposes (using the double digest), then the primer will normally be generated based on the flanking sequences of the plasmid, rather than starting off with the restriction site. The reason is that longer primers ensure a higher fidelity of the product and the fact that polymerases work better with a certain minimum length (routinely 18-20 are used).

 

In the end I do not really understand the question in the OP. It sounds to me that the purpose is a restriction analysis of a vector but why would primer be necessary? In order to sequence a whole vector, depending on its overall size it would be fragmented (which can be done with restriction enzymes) and subcloned into sequencing vectors. One can also do thinks (albeit with lower efficiency) like primer walking. But I do not really believe that that is what is meant.