CRITICALAll spins in this protocol should be at 4 Celsius.
CRITICALDO NOT vortex or shake vigorously, as this will shear genomic DNA and contaminate the prep
CRITICALDO NOT incubate longer than 5 minutes as NaOH will cleave DNA.
CRITICALAlways make Solution 2 fresh to maintain potency!
PAUSEOnce Solution 2 is neutralised, this is an excellent opportunity for coffee or washroom break.
PAUSEPrecipitated plasmid can be safely stored at 4 Celsius for several days.
This wash step replaces isopropanol with ethanol, which is more volatile and therefore will allow the plasmid pellet to dry faster.
As the pellet dries, it will release liquid that can be aspirated to facilitate drying. A dry (-ish) pellet will appear more translucent.
PAUSEDNA is happy and safe in TE. Store at 4 Celsius if required.
PAUSEPEG precipitation is generally better O/N at 4 Celsius, so trade off between time, convenience, and yield.
CRITICALPEG precipitation removes small and unincorporated nucleotide species by specifically precipitating larger nucleotide species, e.g. plasmid.
PAUSEDNA is happy and safe in TE. Store at 4 Celsius if required.
The silicone grease will stabilize the aqueous-organic interface, allowing easier transfer of the aqueous phase without taking junk. The combo of phenol and chloroform denatures and precipitates protein better than either individually. Isoamyl alcohol reduces foaming at the aqueous-organic interface.
CRITICALPhenol is caustic and should be handled with caution. A lab coat is recommended, gloves are absolutely necessary, and all work should be done in the fume hood.
The aqueous layer is usually the top layer, but it can sometimes be the bottom layer if it's denser (i.e. way salty) than the organic layer. You can tell the difference between the two layers by viscosity, i.e. the aqueous layer coheres like water and the organic layer is organic-y. Do not smell to differentiate unless you want to get tipsy and cancer.
PAUSEPrecipitating and precipitated DNA is a good point to pause.
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Old skool purification of plasmid DNA for transfection.