Hi Justin,

The reason you will see increased readings on the Nanodrop in a prep not using chloroform:isoamyl is because the prep will have a significant amount of protein from the bacteria in it.  Amino acids with phenyl groups will absorb light in the same range as the DNA, therefore, over-predicting the amount of DNA.  You are on the right track realizing that the chloroform extracts organic compounds (proteins) from the sample.  Basically, the extraction works as follows-

Alkaline lysis opens up the bacteria and since the bacterial chromosome is attached to the cell wall it will spin down with the rest of the cellular debri (white pellet and white stuff floating after the spin).

The chloroform extraction will cause the proteins to shift into the organic phase and the plasmid DNA will stay in the aqueous phase.  The resulting spin separates the two phases.  

As you know you can then ethanol precipitate the DNA and resuspend.

If you are using a Qiagen or equivalent column then you can skip the organic extraction because the washes of the colunn will remove the proteins.

Hope this helps,

Adam

Hi Justin,

I always use an in house protocol to miniprep preparation. In my case, I worked with 96 well plates and it is too expensive to use commercial kits/columns. If you want to try, I can give you the protocol. But this one does not use chloroform:isoamyl. 

Michele

I think Michele has learnt a lot from experience with mini prep and that is why she has ultimately come out with a feasible methodology which minimises the artifacts like proteins.