Good morning everyone.

I am working on cloning, and this is my first time doing that. I have trouble with cloning now.

I have 4 inserts of DNA.

DNA 1: ~2kb (digested by AscI/StuI)

DNA 2: ~4.4kb (digested by StuI/StuI)

DNA 3: ~6.5kb (digested by StuI/StuI)

DNA 4: ~8.8kb (digested by StuI/BamHI)

My vector has a size of ~11kb (digested by AscI/BamHI)

I did ligase by T4 DNA ligase kit with 225ng of vector and ratio vector:insert=1:3 for each insert DNA.

I incubated the ligation mixture at 14oC for 20h and transformed it to DH10B by electroporation. The condition of the electrical pulse is 25uF, 2.5kV & 100Ω with 0.1cm cuvette.

After incubating the plate overnight at 37oC, I have no colony on my plate. Besides, the positive control plate has colonies.

I also tried different ratios between vector & insert in ligation (1:1, 1:2), but still no colonies on the plate.

What should I do?