Weiss-Lehman Beetles submitted 5-19-2021

Owned by Gregg Randolph
Last updated: Jun 21, 2021 by Shannon Harris

Samples

  1. Transfer beetles to labeled safelock tube

    1. Decant EtOH using filter paper to catch any beetles in the decanting process. Change filter paper if beetles decant. Discard EtOH. (This part should be done by a graduate student from the Weiss-Lehman lab).

  2. Set Heat Block to 56 C

  3. Add single cleaned bead and bead beat in sets of 48 until a Beetle powder is created. (Attempt to centrifuge the first few down although due to the nature of the specimen, the sample may just stick to the tubes).

  4. Add 180 ul PBS with 1250 ul Expandable Pipette

  5. Add 20 ul Proteinase K with 50uL expandable pipette.

  6. Add 200 ul Buffer AL with 1250 ul Expandable Pipette

  7. Vortex for 10s and add to Heat Block for 10 Minutes

  8. Vortex for 10s, then quick spin to drop liquid

  9. Add 8 ul RNase A with 50uL expandable pipette, vortex for 10s

  10. Incubate at RT for 2 minutes, quick spin

  11. Add 200 ul ETOH with 1250ul expandable pipette and vortex 10s

  12. Quick spin (centrifuge), then transfer 600 ul with expandable pipette to labeled spin column

  13. Centrifuge 1m 6000 rcf and discard flow through

  14. Transfer spin column to clean tube and Add 500 ul AW1 with expandable pipette

  15. Centrifuge 1m 6000 rcf and discard flow through

  16. Transfer spin column to clean tube and Add 500 ul AW2 with expandable pipette

  17. Centrifuge for 3 min at 20,000 rcf and discard flow through

  18. Transfer spin column to new labeled and lidded tube

  19. Add 100 ul AE as close to the filter as possible with expandable pipette

  20. Centrifuge for 1 minute at 6000 rcf. Keep FlowThrough

  21. Repeat Steps 18 and 19