I'm attempting to perform a transformation that has succeeded the first time with different oligoduplexes and then failed 4x with different oligoduplexes, which in theory work just fine. Protocol is down below, hopefully someone is able to help me out a bit :)

I'm working with oligonucleotides that I first hybridize to oligoduplexes. The oligoduplexes contain the right overhangs, validated in-silico. Strands are reverse complementary to eachother etc. In-Silico they work. The hybridisation reactionmix contains 1ul 100uM Strand A, 1ul 100uM strand B, 6ul MilliQ and 2ul 5x T4 DNA ligase buffer. PCR protocol is 37C for an hour, 95C for 10 minutes and then cooling down to 25C with 0.1C per second cool down. After that, 5ul is diluted 200x in 1ml MilliQ.

The vector I'm using contains the appropriate overhang. Previous experiment with the same vector but different oligos worked. The vector is cut with BsaI, confirmed on agarose gel (and it worked in previous transformation + positive control).

The ligation consists of 2ul vector (25ng per QuBit dsDNA measurement) (At the first succesful transformation I used a lot less DNA, used nanodrop concentration that was about 30x higher than what the QuBit indicates, used that same concentration for 3 failed transformations), 1ul of the of the diluted oligos, 1ul T4 DNA ligase and 1ul 5x T4 DNA ligase buffer. The ligation is done overnight at 4C, as that is what this lab has found to work best/most convenient and had good results with it.

The transformation is done with Sig10 chemically competent cells, stored in -80C freezer. 15 minutes on ice to thaw, then adding the ligation mixture and flicking the tubes to mix it. Half an hour incubation on ice, then 30 seconds at 42C waterbath and 2 minutes on ice. After that, 800ul recovery medium is added (from the kit) and incubated for an hour at 37C with agitation. 100ul (10%)is then plated on LB medium with appropriate antibiotics. Rest of the liquid is put in a centrifuge, 650ul removed, then resuspended and 100ul is plated again (90%).

What is going wrong here? My first transformation with other oligos worked just fine, did seemingly everything the same. The transformation itself doesn't seem to be the issue due to the positive control always showing. I've tried it 4x now with newly isolated vectors, newly hybridized oligos, new T4 DNA ligase and buffer.