I have a codon-optimized gene cloned into pUC57 with AvrII and XmnI overhangs. I want to subclone the gene into a yeast expression vector (pKOV410). The size of my gene is 1429 bp and the expression vector is 6352 bp. The overhangs are close to each other on the vector. I did a doble digest on the pUC57 to release my gene, ran a gel and extract my gene of interest using Promega gel extraction kit with a final elution volume of 50 ul. I get 20ng/ul concentration of my gene. I also did a double digest of the pKOV410, run and extract the linearised vector. The nanodrop concetration of the vector is 45ng/ul. I am using NEB ligation kit. I tried ligation at different environment (3hr RT, 16hr O/N, 4 degrees O/N). I take at least 3-5 ul of my ligation into 50ul JM109 competent cells which we just bought. I do a control with uncut vector and i get hundreds to thousands of colonies. The ligation kit is new and i made aliquots of the buffer the day i received the kit. The enzymes are from NEB & they recommend digestion for 5-15min, but it seams not to be enough, so i went for 1-3 hours, that works well. But i still dont get a single colony. One of the overhangs is blunt & the other is sticky. I have not tried sequential digest yet. The antibiotic resistance for the expression vector is Kanamycin. I have tried different ligation ratios (1-20) but NO colonies.
ANYONE WITH SUGGESTIONS THAT CAN HELP ME GET PASS THIS STEP?