Annealing oligos with CutSmart

To optimize annealing conditions, I tested the annealing of the following primers-

OligoSim_I-SceI-1_for AGAggtctcaccctCGCTAGGGATAACAGGGTAATGGCtacatgagaccGGC
OligoSim_I-SceI-1_rev GCCggtctcatgtaGCCATTACCCTGTTATCCCTAGCGagggtgagaccTCT
OligoSim_I-SceI-2_for ccctCGCTAGGGATAACAGGGTAATGGC
OligoSim_I-SceI-2_rev tgtaGCCATTACCCTGTTATCCCTAGCG

OligoSim-iSceI-1

 

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.

Plate_28 Plate_27 Plate_26 Plate_25 Plate_24 Plate_23 Plate_22 Plate_21 Plate_20 Plate_19

UPDATE: sequenced OligoSIM and got 100% correct.

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