Rapid Alkaline Extraction of Plasmid DNA
(Adapted from Birnboim, HC and Doyle JJ (1979). A rapid alkaline extraction procedure
for screening recombinant plasmid DNA. Nucleic Acids Research 7: 1513-1523)
1. Grow E. coli cells overnight in 5 ml LB broth and appropriate antibiotic. Patch a
small amount to a fresh LB-antibiotic plate and grow overnight as well.
2. Fill a microfuge tube with the culture and pellet cells by spinning for 1 minute.
Remove supernatant completely.
3. Repeat step 2 as needed. ***Cells can be frozen at this point if needed***
4. Resuspend the cells completely in 100ul LYR by pipetting up & down w/
Pipetman. Incubate 5-10 minutes at room temperature.
5. Add 200ul alkaline SDS, mix by inverting. Mixture should be clear completely
and become very viscous; no clumps should remain. Incubate for 5 minutes a t
room temperature.
6. Add 150ul high salt solution and MIX WELL. The cells debris and chromosomal
DNA will be come a white flocculent material. Incubate 30 minutes on ice
7. Spin in microcentrifuge for 5 minutes. Transfer liquid in to fresh tube, avoiding
floating particles as much as possible (KEEP liquid).
8. Repeat step 7.
9. Precipitate nucleic acid with equal volume (400ul) of isopropanol at room
temperature for 5 minutes. Spin for 5 minutes at room temperature.
10. Remove supernatant, KEEP PELLET. Add 1 ml cold 80% ethanol and vortex.
Spin for 5 minutes.
11. Repeat step 10. Dry pellet at 37°C or in Speed-Vac.
12. Resuspend the pellet in 50ul ddH2O
LYR (50 ml) – store in 1.3 ml aliquots at –20°C
10% glucose
5g
10 mM EDTA pH 8
1.0 ml 0.5 M, ph 8 stock
25 mM Tris pH 8
1.25 ml 1 M pH 8 stock
2 mg/ml lysozyme
100ul
2 mg/ml RNase A
100ul
Dissolve RNAase A in ~ 50 ml ddH2O, boil for 15 minutes to inactivate DNases – being careful
not to boil off all the water then let cool slowly to room temperature. Add remaining components
and q.s to 50 ml.
Total VOL
ALKALINE SDS (make fresh for each prep
1 ml 1 N NAOH (100ul)
0.5 ml 20% SDS (50ul)
8.5 ml ddH2O (850ul)
10
HIGH SALT SOLUTION (5M KOAc)
147.3 g KOAc
115 ml glacial acetic acid
ddH2O to 500 ml