To optimize annealing conditions, I tested the annealing of the following primers-
In different NEB buffers. The following are the plate transformations (plated on chemically competent TG1 cells made with zymo mix and go)
Plate_19) I-SceI_1 primers + NEB 1
Plate_20) I-SceI_1 primers + NEB 1
Plate_21) I-SceI_1 primers+ NEB 1
Plate_22) I-SceI_1 primers +NEB 1
Plate_23) I-SceI_1 primers + Cutsmart
Plate_24) I-SceI_2 primers + NEB 1
Plate_25) I-SceI_2 primers + NEB 2
Plate_26) I-SceI_2 primers + NEB 3
Plate_27) I-SceI_2 primers + NEB 4
Plate_28) I-SceI_2 primers + Cutsmart
All were prepared using a CAS1200 robot with 200ul conductive tips. On attached plates, GFP colonies appear see through, and I also circled them. On some, TG1 was at the side of the tube, and a full 10ul wasn’t plated, and those plates were labeled with an L. It appears Cutsmart and NEB 1 were the most efficient and that I-SceI_2 was more efficient. This is a small test, and nothing was fully verified.
Overall, using bare oligos in cloning reactions looks viable.
UPDATE: sequenced OligoSIM and got 100% correct.