cloning pcr insert into plasmid - (Dec/16/2012 )

Hi,

i am trying to clone a pcr insert of 2kB into a plasmid pYES of 6kB.

i designed the primers (2bp additional nucleotides+NcoI sequence+16bp gene sequence)

the pcr insert is purified,then digested with NcoI (37degC, 3 hours). the plasmid is digested with NcoI too.

after ligation, the ligation mix (10 microL) is electoporated into competent E.coli, all i obtained is many,many,many empty plasmids (without insert)

before, i have tried to dephosphorylate the digested plasmid, but i obtained no colonies on LB ampicillin agar at all

i suspected that it is either the insert is incompletely digested with NcoI or the insert: vector ratio is incorrect (i put 6microL insert:2 microL vector).

any suggestion? is it possible that only adding 2bp additional nucleotides before the RE sequence caused the incomplete digestion?

thanks

Yes, it is possible that 2 bp is too few. However, NEB says that 2 should be enough... Try using an excess of RE relative to amount of DNA. You do need to dephosphorylate your vector.

DNA ratios for cloning need to be molar ratios. Calculated as follows: ng insert = ratio x x ng vector.

Make sure that the amount of ligation mix used in the transformation is less than 5% of the volume of the transformation volume.

thank u very much

i hope it works this time...