Sticky end cloning

This is Alan's protocol to aid the efficiency of successful sticky end cloning.

Insert preparation

PCR up the fragment you want to clone (either from a plasmid or from genomic DNA)

Gel extract the amplified fragment

Cut with restriction enzymes

Carry out a PCR cleanup reaction (Alan adds isopropanol is the fragment is greater than 4kb or less than 500bp as this is supposed to aid binding to the silica in the column)

Vector preparation

Cut the vector with restriction enzymes

Gel extract the cut vector (run uncut vector on the same gel if you want to be certain which band is which)

Next stage

If both cut insert and vector are run on the same gel you can use this to quantitate the amount of DNA required for cloning (a 3kb vector and a 1kb insert in 1:3 molar ratio will be about the same intensity on a gel). However, it's probably more accurate to use the nanodrop and measure the [DNA] this way.

Then proceed to ligation. If you want to get an idea of the amount of uncut vector / vector religation, run the following controls:

• vector + insert

• vector only + ligase (control for religation)

• vector only, no ligase (control uncut vector)

To test whether the correct insert has been ligated into a particular colony, carry out PCR screening.