Hi, I'm trying to perform a Gateway Cloning procedure. I have tried to clone a PCR Product into an entry vector via TOPO-Cloning, but, according to my gel electrophoresis it is not working. Here is an overview of what I have done:
Day 1: 3.3kb sequence amplified via PCR Product --> checked with gel, worked.
Day 2: Gel was extracted and DNA isolated via Qiagen Gel Extraction Kit --> measured sufficient amount of DNA, proceeded to TOPO-Reaction
Day 3: Performed TOPO Reaction via pENTR-DONR TOPO Cloning Kit (pENTR-DONR Vector = 2.5 kb) --> transferred TOPO mixture to One-Shot E. Coli cells and plated on selective LB agar (antibiotic = kanamycin), cultured overnight
Day 4: Obtained colonies --> assumed these bacteria had transformed TOPO vector, since the TOPO vector had kanamycin resistance. --> Picked up colonies and transferred to Kanamycin-LB broth to grow for 16 hours.
Day 5: Pelleted cells and isolated DNA via Qiagen MiniPrep DNA Extraction Kit --> measured plasmid DNA to be 146.95ng/uL.
I made sure that the NanoDrop machine was measuring DNA, and when the DNA sample was run on a gel, there was no band detected at all. I did Day 4 (regrew cells) and Day 5 (pellet + MiniPrep) twice more, and still couldn't get a band. Theoretically I should be seeing a 5.8kb sequence but there was nothing. Could it have been contaminant DNA I was measuring? Even if it was there was it would have detected by the gel.
I also added EtBr to the agarose gel and dye to the sample before loading the sample into the gel well. Should I be adding something to the DNA sample to detect the band?
I also increased my DNA loading from 2uL to 4uL, and there was still no detection. Should I increase my loading amount? Or can someone give me suggestions? Thank you!