Genomic DNA maxiprep

DNA extraction

Important notes
•Start with a frozen pellet of 80 x 10^6 cells for QIAamp DNA Blood Maxi Kit
• Follow manufacturer’s instructions
• Use only gDNA prep-designated sterile water and ethanol stocks
• To avoid contamination do not bring whole sample to Nanodrop
• Store gDNA and PCR reagents SEPARATELY from PCR and bead cleanup products

Day one
1. Warm water bath to 70C
2. Make 0.5ml proteinase-K + 9.5ml PBS solution per sample
3. Add to cell pellet and mix
4. Add 12ml Buffer AL and mix THOROUGHLY ( 15 sec vortexing) until
the mix is white (from the amount of bubbles)
5. Incubate 70ºC overnight (recommended) OR 1.5hr with brief mixing at regular intervals

Day two
6. Add 10ml 100% EtOH and mix thoroughly by inverting.
7. Pipette 1/2 volume onto DNA maxi column and spin 3min @ 3,000 RPM (after
spins check membrane: it should be dry - if not, spin longer)
8. Discard flow-through and repeat with other 1/2 of mix.
9. Add 5ml Buffer AW1 and spin 2min @ 5,000 (we use 4,000), no need to
discard at this step
10. Add 5ml Buffer AW2 and spin 15min @ 4,000 RPM
11. Place in the collection tube, add 1ml H2O
12. Spin 5min @ 3,800 RPM
13. Re-apply elute to the column and let the sample sit in 4C for 30min-2hr
14. Spin 5min @ 3,800 RPM Nanodrop a small aliquot of the sample.
15. Heat 95C 5min.
16. Proceed with test PCR.