McClean:Genomic DNA prep (LiAc SDS method)

Overview

This protocol is for rapidly isolating genomic DNA from an yeast colonies. These protocols were adapted from Lõoke M, et al. (2011). Please see the reference below and cite it if you use this protocol.

Materials

• Yeast colonies
• 0.2 M LiAc 1% SDS solution
• 100% Ethanol
• 70% Ethanol

Protocol (tubes)

1. Pick a colony into 50 uL 0.2 M LiAc 1% SDS solution and mix well by vortexing
2. Incubate at 70C, 5 min.
3. Add 150 uL 100% EtOH
4. Centrifuge at 14,000 rpm, 30 sec, room temperature
5. Aspirate the supernatant
6. Resuspend in pellet in 100 uL 70% EtOH
7. Pellet and aspirate as before
8. Resuspend pellet in 25 uL H2O
9. Centrifuge at 14,000 rpm, 30 sec, room temperature to collect cell debris
10. Use 1 uL supernatant for PCR

Protocol (96-well plates)

1. Pick a colony into 50 uL 0.2M LiAc 1% SDS solution in a 96-well PCR plate
2. Heat to 70C, 5 min
3. Add 150 uL 100% EtOH
4. Centrifuge at 3,000 rpm, 5 min
5. Drain the supernatant onto paper towels, about 5 minutes
6. Resuspend pellets in 70% EtOH
7. Pellet and drain as before
8. Resuspend pellet in 25 uL H2O
9. Centrifuge at 3,000 rpm, 5 min to collect cell debris
10. Use 1 uL supernatant for PCR

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

• Taylor D. Scott (talk): This protocol works equally well in 96-well plates and in tubes in my experience.
• Taylor D. Scott (talk): PCR from this gDNA works well for Sanger sequencing.
• Taylor D. Scott (talk): I've changed the centrifugation times to 30 sec (tubes) and 5 min (plate). I notice no difference in efficacy using the lower spin times.

References

• Lõoke M, Kristjuhan K, and Kristjuhan A. "Extraction of genomic DNA from yeasts for PCR-based applications." Biotechniques. 2011 May; 50(5): 325–328. doi: 10.2144/000113672.

Contact

• Taylor D. Scott (talk):

or instead, discuss this protocol.