Hi..

My vector is pGEM-T cloning vector (3kb) and my insert size is 1992 kb (~2kb). I use AgeI and KpnI for double digestion. For the competent cells, i use DH5a to clone the plasmid.

I did overnight digestion and also 3 hours digestion by using master mix.

For both experiments, I got partial digestion for AgeI and possibility for KpnI having Star activity when doing single digestion

However, when I did double digestion, I got two bands, and the lowest band represent my insert size. Seems that my REs are working in double digestion.

Next, instead of using master mix, I directly mix the reagents and incubate for 2 hours. For KpnI, i use 0.5 uL per rxn vol.

There are no multiple bands & star activity for KpnI..But now both KpnI and AgeI show incomplete/no digestion

AND my double digestion looks fine..

My question is, 

Do the AgeI and KpnI are really working?

What can I do to solve incomplete/no digestion without having to increase the incubation time because I'm afraid it will trigger KpnI star activity?

Thanks for your responses :)