Hey ho,

I have a really hard time with cloning right now and happy for any advise.

I am using E. coli NEB5alpha and pET28a vector. The vector is PCR-amplified plus DpnI digested or digested with restriction enzymes NheI and XhoI. Both constructs results in a nice band on a gel and no colonies for my negative control.

For ligation, I am using the Gibson assembly for 15 min at 50°C. My insert is about 3000bp long, while my negative control is only water plus vector.

So as I said, there are no colonies for my negative control and >30 colonies for my insert, but my colony PCR results in only a band of about 300 bp (for the multiple cloning site) but not for the size of my insert. I have screened about 15 colonies now.

My PCR protocol accoding to NEB:

12.5 µL 2x Quick load master mix

1µl template ( I mix a tiny bit of a colony in 50 µL)

0.5 µL 10µM T7 forward primer

0.5 µL 10µM T7 reverseprimer

10.5 µl water

95°C 5 minutes

30 cycles of:

95°C 30 sec

45°C 1 minute

68°C 4 minutes (1minute/kbp)

68°C 5 minutes final extension

I hope anyone has suggestions. I have changed all reagents for PCR and my insert and vector are purified and the concentraion is fine. Maybe I miss / do something (obvious) wrong.

Thanks in advance!