SOP for GWAS Fecal Collection and DNA Extraction for Downstream Sequencing Applications

 

 

Materials:

 

                                Denville Posi-Click Tubes 1.5ml        500pk                       C2170                                   

Quick-DNA Fecal/Soil MiniPrep Kit   50pk                        D6010  

                                

 

Protocol for Feces Collection:

 

  • Collect fresh feces – this must be directly from the animal. Dried feces from the night before, or in the cage will not yield good DNA for sequencing.

  • Take approximately two small piece of fresh feces by gently holding the rat by the tail with the front paws on a cart and bounce the animal so the hind legs come gently off the cart – this will make the animal defecate in 30sec-1min.

  • Place the fresh feces into a marked tube and immediately put in powdered dry ice until it can be transferred to the -80 where it will be stored.

 

 

Protocol for DNA Preparation:

 

  • Add up to 150mg fecal sample to ZR Bashing Bead Lysis Tube

  • Add 750ul Lysis Solution to the tube

  • Secure in a bead Beater fitted with 2ml holder assembly and process at max speed for 5 min.

  • Centrifuge for 10K for 1min

  • Transfer 400ul Supernatant to Zymo Spin IV Filter in a collection tube and centrifuge for 7K rpm

  • Add 1.2ml Fecal DNA Binding Buffer to the filtrate in the collection Tube

  • Transfer 800ul of mixture from step 6 to Zymo-Spin IIC column in a new collection tube and centrifuge at 10K for 1min.

  • Discard flow-though from step 7 and repeat.

  • Add 200ul DNA Pre-wash Buffer to the Zymo-Spin IIC column in a new collection tube and centrifuge at 10K for 1 min.

  •  Add 500ul Fecal DNA Wash Buffer to the Zymo-spin IIC column and centrifuge at 10K for 1 min.

  •  Transfer Zymo-Spin IIC column to a clean 1.7ml centrifuge tube (Denville Posi click) and add 100ul DNA Elution Buffer directly to column. Centrifuge at 10K for 30 seconds.

  •  Transfer eluted DNA from step 10 to prepared Zymo-spin IV HRC filter  - into a clean 1.7ml centrifuge tube and centrifuge exactly 8,000g for 1 min.

  • The filtered DNA is now suitable for PCR and downstream applications.

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