Biol 420 Fall 2007
(as in Manual ArchivePure DNA Purication by 5 Prime)
Put 300 ul of lysis solution into 1.5 ml microfuge tube.
Brush inside of check with at least 10 strokes with a sterile brush
Insert into the 1.5 ml microfuge tube containing the 300 ul of lysis solution
Swish around for approximately 1 minute
Cut off tip with sterile blade or scrissors and leave in the 1.5 ul microfuge tube and label microfuge tube(s)
Add 1.5 ul of Proteinase K solution (20 mg/ml) to cell lysate, mix by inverting 25 times and incubate at 55 ۫ C for 1 hour.
After the hour has elapsed remove brush making sure to scrape the brush along side of the tube to remove any excess lysate from the collection brush head
Add 1.5 ul of RNase A solution to the tube, invert 25 times, and incubate for 20 minutes at 37 ۫C, once the 20 minutes has elapsed place in an ice bath for 1 minute to bring the sample back down to room temperature.
Add 100 ul of Protein Precipitation Solution to the cell lysate
Vortex at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate.
Place tube in ice bath for 5 minutes
After the 5 minutes centrifuge at 14000 RPM for 3 minutes. Make sure the proteins have formed a tight, white pellet. If it is not tight repeat steps 10,11 and 12.
Pour the supernatant containing the DNA into a clean 1.5 ml microfuge tube containing 300 ul 100% Isopropanol and 0.5ul (20 mg/ml stock) Glycogen Solution.
Invert 50 times, incubate at room temperature for 5 minutes
Once the 5 minutes are completed centrifuge at 14000 RPM for 5 minutes. DNA may be visible as a small white pellet.
Pour off supernatant and drain tube on clean absorbent paper. Add 300 ul of 70% Ethanol and invert tube several times to wash DNA.
Centrifuge at 14000 RPM for 1 minute. Carefully pour off the ethanol and allow to dry for at least 15 minutes
Add 20 ul of DNA Hydration Solution overnight at room temperature then store at -4 ۫ C
Start with 2 ul of genomic DNA (then add)
10 ul 10X PCR buffer (+MgCl2)
2 ul (200uM) dNTPS (10 mM)
1.9 ul (0.3 uM) forward primer
1.5 ul (0.3 uM) reverse primer