How do you choose which restriction enzymes to use when cloning a gene?

1 Answer
Nov 8, 2017

Answer:

Depends on the vector you will be using, and the fragment you want to clone.

Explanation:

First, you have to select the best vector to clone into.
In my days, we either used pBR322 (a plasmid), M13, or #phi X#174

Whatever vector you use, it will have a few optimal Insertion Points to clone your fragment into and will define the RE you have to use.

Mind you, I left the cloning business late 80's and time has moved on no doubt, but Nature doesn't change...

You splice the vector with the ER of you choice (like EcoR1 or Hin DIII in the case of the Vectors mentioned above), splice the DNA to be tested with the same RE, Heatshock both samples, purify, combine, and combine wit DNA Ligase. Purify again, and amplify by using the Klenow Fragment of DNA Polymerase 1 (from E.Coli)

Because the Vector Genome is known (M13, #phi X#174 and pBR322 have been well documented in the 70's), you will know where the insertion point is, and Sequencing will be no problem...