Sample Preparation for DNA Methylation Assays

Customers may submit either cells or DNA for Genpathway’s meDNA IP and Bisulfite Sequencing Assays.

MeDNA IP
(Use one of the following recommendations for sample submission.)

     I.   Prepare frozen pellets from cell cultures

1. Grow approximately 5 x 10⁶ cells in culture.
   
2. Transfer cell culture to a conical tube (If cells are adherent, scrape them thoroughly from the culture surface prior to transferring to a conical tube).
   
3. Centrifuge tubes at 800 x g in a refrigerated centrifuge for 5 min to pellet the cells. Decant culture media.
   
4. Re-suspend cells in 10 ml chilled PBS per tube by pipetting up and down and spin again at 800 x g in a refrigerated centrifuge for 5 min to pellet the cells.
   
5. Decant PBS, freeze cell pellets on dry ice, and store at -80°C.
   
6. Ship on dry ice.
   

    II.   Freeze animal tissue

1. Remove appropriate amount of tissue from animal (approximately 100-200 mg for most tissues).
   
2. Place tissue in a 1.5 ml microfuge tube or 15 ml conical, freeze on dry ice, and store at -80°C.
   
3. Ship on dry ice.
   

    III.   Prepare DNA

1. Prepare genomic DNA from cell culture or animal tissue using Zymo Research Quick-gDNA™ MidiPrep Kit or other genomic DNA isolation method.
   
2. Elute or resuspend DNA in water and store at -20°C.
   
3. Run 200 ng of DNA on a 1% agarose gel to show high molecular weight DNA (Figure 1).
   
4. Send 30 ug of DNA at a minimum concentration of 200 ng/ul in water and a photo of the gel analysis.
   
5. Ship at -20°C.
   
   Figure 1.
   
   

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Bisulfite Sequencing
(Use one of the following recommendations for sample submission.)

     I.   Prepare frozen pellets from cell cultures

1. Grow 5 x 10⁵ to 2 x 10⁶ cells in culture.
   
2. Transfer cell culture to a conical tube (If cells are adherent, scrape them thoroughly from the culture surface prior to transferring to a conical tube).
   
3. Centrifuge tubes at 800 x g in a refrigerated centrifuge for 5 min to pellet the cells. Decant culture media.
   
4. Re-suspend cells in 10 ml chilled PBS per tube by pipetting up and down and spin again at 800 x g in a refrigerated centrifuge for 5 min to pellet the cells.
   
5. Decant PBS, freeze cell pellets on dry ice, and store at -80°C.
   
6. Ship on dry ice.
   

    II.   Freeze animal tissue

1. Remove appropriate amount of tissue from animal (approximately 1-5 mg for most tissues).
   
2. Place tissue in a 1.5 ml microfuge tube, freeze on dry ice, and store at -80°C.
   
3. Ship on dry ice.
   

    III.   Prepare DNA

1. Prepare genomic DNA from cell culture or animal tissue using Zymo Research Quick-gDNA™ MidiPrep Kit or other genomic DNA isolation method.
   
2. Elute or resuspend DNA in water and store at -20°C.
   
3. Run 200 ng of DNA on a 1% agarose gel to show high molecular weight DNA (Figure 1).
   
4. Send 1-2 ug of DNA at a minimum concentration of 25 ng/ul in water and a photo of the gel analysis.
   
5. Ship at -20°C.