Isolation of plasmid DNA - alkaline lysis method (Nov/22/2006 )

anyone know why after the addition of STET buffer and lysozyme, i would pierce the lid of microfuge and boil??

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i'vfe never do that.

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I have never used the STET protocol..... however by reading it I can make a guess

"piercing the lid of microfuge tube" is the standard practice when you want to boil something in said tube. The hole in the lid is to relief excess pressure. If you don't put a hole in the lid, then the tube is very likely to "pop" during the boil or just after taking it off .... the lid pops off, throwing the contents of the tube everywhere. It is a bit messy, and with several poping events going on at the sametime, likely to cross contaminate.

The boil in the STET protocol is probably to help lyse the bacteria cells (thermal lysis), and to denature the cellular proteins, which also probably help pull down genomic DNA.

If my understanding is correct, is very important to use a large volume of STET buffer (350ul - 500ul) as lysozyme maximum activity is at 50 Celsius and denatures at 75 Cesius. The bulk volume, helps decrease the rate of heating, giving the enzyme more time to degrade the peptidoglycans of the bacteria cell wall.

Perhapas the protocol could be improved by heating at 50 Celsius for a short while (maybe 2-5min) before ramping up the temperature to a boil? I wonder if that will work?

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i use alkaline lysis method to prepare plasmid DNA. but i never boil my sample after adding STE

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