Hi,

I have been attempting to clone a 48bp insert into a 11,200bp vector. The insert is formed from two single stranded oligos, which when annealed should form AAAA and TAAT sticky ends. I have been annealing the two by mixing in a 1:1 ratio in a PCR tube and heating it to 98C for 2 mins and 55C for 5 mins. Gel images show the annealed product higher than the two ss oligos.

BsaI is used to open the vector and expose complementary sticky ends TTTT and ATTA. I then use T4 ligase to ligate the product. This is done overnight at RT. product is used for transformation in E.coli. After many attempts resulting in no colonies on the ligated plate, I increased the vector insert ratio to 10:1. This resulted in 8 colonies. However, by checking on a gel with primers designed to amplify if the inserted fragment is present, it is clear that ligation was not successful as multiple products have formed at strange heights.

Should I be phosphorylating the annealed insert with PNK? I have read that this shouldn't matter since a phosphate is present in the vector when cutting with BsaI. Is there any other key aspects I am missing to make this successful?

Thanks for any help!