Week 13. Semester 2. Still no DNA but plenty of fungus!!

This week I attempted another DNA extraction with the protocol under development to be added to Protocol 17 (DeSalle 2002). The following was done:
1. Prepared citrate buffer and diluted to 50mM (Adney 1996).
2. Added 50microL Sigma Cellulase from aspergillus solution (1000U/g)
3. Used +/- .450 g of leaft tissue and the same from blossoms in simultaneous runs.
4. One set of samples was ground with mortar and pestle with buffer until paste was formed. The other half was added to buffer and shook until immersed in citrate buffer/enzyme solution in bottom of tube.
5. Incubated at 30C for 24 hours.
6. Centrifuged at 12,000rpm for 5 min.
7. Transferred supernatant to new tubes and added 500 microL of lysis buffer (3:1 4% SDS to 5% Sarkosyl).
8 Inverted to mix.
9. Incubated 5 min.
Proceeded with Protocol 17.

The DNA in solution was electrophoresed in 1% agar gels for 30 min at 100V and then viewed under UV light. The picture below shows no visible DNA. The bottom bands are from control from a previous extraction.

I also added some pictures of my fungal isolations.

References

     Adney B and Baker J. 1996. Measurements of cellulase activities: Laboratory analytical procedure. Golden (CO): National Renewable Laboratory; p. 2.

 

     DeSalle R, Giribet G, Wheeler W. 2002. Protocol 17. In: Techniques in molecular systematics and evolution [Internet]. Springer science and business media. p. 276-278.