To prime efficiently, double-stranded plasmids must be converted to single-stranded form prior to sequencing. This is accomplished by alkali denaturation of supercoiled plasmid DNA.
The plasmid pellet left from section miniprep was resuspended in 18
l water. 2 l of a 2M NaOH, 2mM EDTA solution were added and incubated
for five minutes at room temperature. The reaction was neutralized with 2
l of 2M ammonium acetate (pH 4.6) and mixed. 75 l of ethanol were
added, mixed and precipitated for 10 minutes at -70psy176 C. The reaction was
now centrifuged for 10 minutes at top speed, the supernatant decanted, the
pellet washed with 100 l cold 70% ethanol and centrifuged for one minute.
The supernatant was decanted and the pellet vacuum dried (Speed Vac).
The remaining pellet can be stored at -20psy176 C and is resuspended in 18
l water for sequencing.