Even if there is a polylinker, then so what?

[Genecks]

So, let's say I have this:

pUC18 vector: http://www.lgcstandards-atcc.org/attachments/1477.jpg

 

What does it matter that the polylinker is in there?

Isn't there the probability that the cut sites will already be present in other sections of the plasmid?

 

Or this kind of vector such a standard that the enzyme restriction sites won't be found anywhere else but in the polylinker, thus creating a confined place to put the insertion?

[Greippi]

The restriction sites in the polylinker are found nowhere else in the plasmid. The poly linker is right in the middle of the lacZ gene, this placement is important in selecting colonies which are expressing your gene:

 

Expression can be initiated by the addition of IPTG in the growth medium as your gene is now under the control of the lacZ promoter.

If your gene is NOT correctly inserted in to the plasmid, beta galactosidose is encoded by lacZ and produces blue colonies.

If your gene is inserted in the plasmid, it interrupts the lacZ gene meaning that beta galactosidase is not produced and the colonies appear white.

 

So you know white colonies contain your gene, and you can select for those.

[CharonY]

MCS are necessary to a) place the gene in a specific area, which is important for subsequent analyses as e.g. sequencing reactions, b) provide a large number of unique enzyme cutting sites to provide versatility to your cloning reaction and c) as Greippi mentioned, allow counter selection.

 

Expression can be initiated by the addition of IPTG in the growth medium as your gene is now under the control of the lacZ promoter.

This may happen, but with puc18 vectors you try to gene expression. As the MCS is not "flush" with the ORF, either nothing or garbage gets produced. If you really want expression you go for (over)expression plasmids.

 

Without an MCS you are very limited in the way you have to create your fragments.