Plasmid recovery problem
Posted 26 January 2012 - 01:19 PM
I'm having major issues with plasmid minipreps. I've grown my culture from glycerol stocks and the overnight culture gives me a very dense culture (5 mL). I've spun this down and did my miniprep using the Invitrogen kit and I'm getting very very low concentrations (~35 - 70 ng/ul). I thought maybe the kit was old, so I got a new and I'm getting the same results. I have no idea why this is happening.
Posted 26 January 2012 - 02:19 PM
Posted 27 January 2012 - 07:16 AM
Check your wash buffer. I had the same low-yield problem with an old miniprep kit. What had happened was the ethanol evaporated away, leaving the buffer. It still smelled like ethanol, but enough had evaporated to wash all my DNA from the column during the wash step.
Make the wash buffer fresh. A homemade prep of 80% ethanol (final conc) diluted in 10 mM Tris, pH 8 will work for most commercial kits. The ethanol concentration in the wash HAS to be at least 70%.
If you still have low yield, you may have a low copy number plasmid. Grow several 5 ml cultures overnight, isolate plasmid DNA in separate columns and pool the final eluates. A quick ethanol precipitation and resuspension in a smaller volume should boost your DNA concentration.