Miniprep yield for pET28 plasmid: 2.5ug. Too low? - Why such a low yield? (Mar/05/2010 )
I am using the Qiagen kit for Miniprep of pET28 plasmid (with an insert). I typically get 50ul of eluted DNA with conc. in the range of 49-70ng/ul.
Is this yield too low? The manual says that typical yields are in the range of 100-200ug DNA per prep for high copy no. plasmids. Where could I be making a mistake?
I use LyseBlue and everything seems to work well.
For a standard miniprep, your yield seems typical. I think you must be reading a midiprep or maxiprep protocol, which would start with much higher amounts of culture medium.
I agree with phage. Concentrations for pET-28 usually run 45 - 100 ng/ul with Qiagen minipreps.
We've been able to get around 100 - 250 ng/ul pretty consistently.
There are a few things you can do if you run into problems if your concentrations are too low for sequencing or if you're not getting a lot of colonies after transformations:
1) Reduce your elution volume to 30 ul if still too low, try 15 ul (you may need to do a few preps to get the volume that you will need for sequencing, transformations, and storage stock.)
2) Reduce the amount of supernatant applied to spin column, making sure to only apply clean solution without cell debris of any kind - really - sometimes the column is overloaded or gets gunked up and your concentrations are reduced as a result.
3) Warm your elution buffer in a 37C - 45C water bath just prior to elution
The concentration you are getting is typical for miniprep. You can either reprecipitate your DNA with Ethanol and suspend in smaller volume or maxiprep as other guys suggested.