troubleshooting + ligation

[Anajli Thomas]

      I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The
restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice
and still no colonies. JM109 was used for transformation.

The restriction enzymes were added together for cutting the insert from T
vector (primary clone). For ligation I added 5 ul of insert and 2ul of
backbone.

Could you help me out with this problem

Sincerely
Anjali