Sunday 28 June 2009

molecular biology - What is a good miniprep protocol for the class room?

I think the spin column kits are the way to go. As mentioned already, the benefits of the kits are that they are easy, safe, and (most importantly) how almost every actual lab does plasmid purification these days.



The biggest criticism of the commercial kits is that you can get by easily without knowing anything about what is actually happening in the tube. However, as noted in the comments to Nick T's answer, there is no secret proprietary technology in the kits--they use the alkaline lysis method. This means that you can still teach the mechanism in the class while getting the benefit of the spin columns.



One option, for teaching purposes, is to make all of your own solutions and just use the spin columns on the supernatant after pelleting the precipitated membrane/protein. That way, you get the benefit of insisting on real names for the solutions (NaOH is a more educational name than P2) and you get to use the spin columns in place of the ethanol precipitation. I believe there are cheap sources that just sell the spin columns, although I have never used them. You want to avoid the ethanol precipitation because it adds a bare minimum of 30 minutes to the protocol (for my protocols, it was more like an 1.5 hours or more). And waiting for ethanol to evaporate so you can resuspend is like watching paint dry unless you have a speed vac.



Yield shouldn't be a big issue in a class situation, but you can boost yield with the column by prolonging the final elution step. I think the given instruction is to let the TE buffer (Tris) "elute" for 1 minute before the final spin, but I have routinely let the TE sit for about 15-30 minutes before spinning down. The difference in yield blew out the exposure on the gel camera.



There are certain cases where phenol:chloroform extractions are still desirable, but this is definitely not one of them. I used to do P:Cs routinely when working with RNA and yeast genomic DNA. I have never seen anyone do anything other than a miniprep when preparing plasmids. What's more, the hassle of working in a fume hood and the risk of phenol burns encouraged a lot of people in the lab to prefer the spin kits even when working with genomic DNA.

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