Hello everybody,

I am currently trying to ligate a _{80 bp duplex synthetic oligo (that has the 2 required restiction site over hangs) into a double digested 5.2 Kb plasmid vector using NheI and SacI. So far I have tried:}

1. 3 different T4 ligases!
2. Postive control that I know works (I have used the same vector and different insert (35 bp duplex) that I had cloned before).
3. After restriction digestion of the plasmid, I have tried both gel purification and without gel purification (tried eliminating the ~60 bp unwanted sequence by Microcon spin down; which should eliminate anything less than 200 bp according to the product catalog)
4. Apart from the 3x molar conc of the insert, I have also tried 10x,20x an 100x insert to vector ratio after reading some posts online…
5. Tried diffrent DH5a batches, and new ampicillin plates.

Even after all that, I have not gotten a single colony with my insert! Also, The synthetic oligo sequence has passed the HPLC quality control of the company and so, I think it is probably not the insert sequence issue.

I have spent about 2 months trying to do all this! I am completely bewildered and not sure what my next step should be…

I would appreciate any thoughts/suggestions that you may have.

Thanks in advance.