DNA disappeared after ligation - (Oct/09/2007 )

I have completely digested vector and insert DNA with ApaI and EcoRI. Vector is from T-vector and insert from one plasmid of my lab. And extracted the fragments by agrose gel.
I think there is no problem with the digestion and extraction, I checked DNA concentration by gel after extraction, the concentration seems good, and they are around 20-30ng/ul.
And I calculated the DNA ends ratio according to this website
http://www.fermentas.com/reviewer/app?page...p;sp=Sligations

I used Invitrogen T4 DNA ligase, 14 degree O/N. then did heat shock transformation, the competent cell is DH5a, the result is no any clone. I tried many insert vector ratio and all failed finally.

so I checked my ligation reactions after ligation by gel, It is weird I can not see any DNA on gel after ligation, I run insert DNA, vector DNA and DNA ladder as control, they all seems good, I am sure the DNA amount I added in the ligation, it is more than 50ng, it can not believe that I can not see anything on gel after ligation. Where is my DNA after ligation? It is my question.

And I also did a TA clone ligation, I can see the higher band than insert and vector on gel after ligation and can get pretty good clones on plate. so why I can not get any DNA band after ligation that I got vector myself not from kit. Even I lost DNA I added into the ligation reaction.

I will appreciate your help. Thanks!

-Beth-

I've found it essential to heat kill the ligase before running ligation product gels. Otherwise, the ligase binds the DNA and you have a gel shift smear, which with only 50ng may well appear as if there is no DNA.

-phage434-