5 replies to this topic

[mahsa]

hey all
is there a database through which one can find vectors solely based on their respective  restriction site? dose any one know a bacterial plasmid which lakes restriction sites for AclI or BglII?

any hint or idea is deeply appreciated

[kajmak]

You can always add these sites in vector you allready have. Just cut the vetor with any enzyme and ligate with oligos containing desired restriction sites. Just make sure they are complemetary to each other (make, say, 30 nt long dsDNA) and phosphorylated.

[mahsa]

kajmak, on 27 August 2010 - 10:51 PM, said:

You can always add these sites in vector you allready have. Just cut the vetor with any enzyme and ligate with oligos containing desired restriction sites. Just make sure they are complemetary to each other (make, say, 30 nt long dsDNA) and phosphorylated.

yea, sure, site directed mutagenesis is almost always handy in a molecular-biology lab, what i meant was what if site directed mutagenesis is not an option. is there, say, a holistic database which you can choose your vector based on different specifications?
tnx for the reply by the way

[phage434]

Short answer: no, there is no such database that I am aware of.  Longer answer: the reason is that it is easy to make a vector with the cloning site you need by PCR, by making primers that amplify the vector origin and resistance, and leave 5' overhangs for the cloning site you want.  You need to be careful that those sites don't already exist on the vector and to leave sufficient 5' overhang past the restriction site so that the enzyme will cut.  PCR, purify, cut (at the same time as your insert is cutting), mix, ligate, go.

[perneseblue]

Nope. It would have been a good idea maybe a decade and a half ago.

But now primers are cheap. Proof reading DNA polymerases are good and getting better. The need and desire has gone. It is expensive ordering in new plasmid vectors and it takes time to transform and prepare the new vector.

Far easier to PCR amplify and modify the plasmid vector that you have sitting the fridge.

[mahsa]

perneseblue, on 30 August 2010 - 08:25 PM, said:

Nope. It would have been a good idea maybe a decade and a half ago.

But now primers are cheap. Proof reading DNA polymerases are good and getting better. The need and desire has gone. It is expensive ordering in new plasmid vectors and it takes time to transform and prepare the new vector.

Far easier to PCR amplify and modify the plasmid vector that you have sitting the fridge.

tnx alot