I’m encountering issues while isolating and visualizing plasmid DNA from E. coli. Even after running the gel, I observe either a smear or very faint/unclear bands. I loaded around 7 µL (2667ng/µL)of my sample. The OD readings seem acceptable, but the gel doesn't show clear plasmid bands.
Could anyone suggest what might be going wrong?
Is there an issue with plasmid concentration or quality?
Could overloading or degraded DNA cause this?
How can I troubleshoot step-by-step to ensure intact plasmid isolation and proper visualization?
Any suggestions, protocols, or similar experiences would be highly appreciated. Thanks in advance!
If your concentration is accurate, then you would have very bright fluorescence bands. My guess is your OD is caused mostly by protein. All that extra gunk in the sample will cause you bands to smear and cause fuzzy results. Did you measure the 260/280 ratio? Ideal is 1.8, but a little lower would be ok. If your ratio is really low, like 1.0 or lower, I suggest phenol/chloroform extracting your prep, add 1/10 volume of 3/5 M KOAc, 2 volumes of EtOH, chill spill, dry and resuspend in TE.