Monday, January 12, 2009

Community Service (better than jail time)

As I often want to make a small amount of a given lentivirus, I find it a pain in the butt to do MaxiPreps to get good, transfection-quality DNA -- i.e., why spend over an hour prepping a miligram of plasmid when I only need about a microgram?

So, I've been using Macherey-Nagel's Nucleobond PC20 preps when I need to do a dozen or so transfection-quality minipreps (quite analagous to Qiagen's transfection-quality mini prep kit). But, this protocol isn't very fast (about an hour, with some standing-around time), and at ~$4 a prep, not particularly cheap.

Promega has a new product out, and since they sent me a free sample, I gave it a try. Their PureYield mini-prep really impressed me. First, it is quite fast, faster than even a standard Buffer 1, 2, 3 miniprep. Second, it costs just over $1 per prep. And most importantly, the DNA performs really well in making virus.

I divided an overnight culture into 3 parts, all in duplicate, of a luciferase-expressing lentivirus.
1) Macherey-Nagel NucleoSpin (i.e. a standard silica mini-prep)
2) Macherey-Nagel Nucleobond PC20 (anion exchange)
3) Promega PureYield

In terms of yield, all three were quite comparable, giving 3.8, 4.1, and 3.5 ug of plasmid from 3 mL of culture, respectively.

I then made virus and infected cells, assaying for luciferase the next day. Both the Nucleobond PC20 & the PureYield gave ~20% more luciferase activity compared to the standard mini-prep. Given that PureYield is 4x cheaper and 4x faster, it is my new mini-prep.