DNA

Soltis Lab CTAB DNA Extraction Protocol

(Reference: Doyle & Doyle, 1987; and Cullings 1992)

Revised for fresh tissue, November 27, 2013

  1. Prepare CTAB buffer, use within 2-3 days, store capped: Add polyvinylpyrrolidone (Fisher Cat#: BP431-500) and ß-mercaptoethanol (Fisher Cat#: BPI 76-100) and stir to dissolve right before starting extractions: CTAB PVP b-merc

                            5 ml                 0.2 g                 25

                            20 ml              0.8 g               100 pi

  1. Weigh out 10-20 mg of fresh plant tissue.
  2. Place tissue with 5 Zirconia Beads (2.0 mm, Biospec) in a 2 ml eppendorf and close tube, grind:
    1. Flash freeze containing beads and tissue in liquid nitrogen.
    2. Cool down the plastic bead mill eppendorf rack in liquid nitrogen.
    3. Transfer the frozen tubes to the cold rack and immediately grind the tissue in the bead mill for 30 sec.
  3. Add 850 gl of CTAB buffer and grind samples a bit more.
  4. Incubate samples at 550 C for 1 hr.
  5. Add 850 gl of 24:1 Chloroform:lso Amyl Alcohol and mix well by shaking tubes.
  6. Centrifuge for 10 minutes at maximum speed.

I, Following centrifugation, you should have three layers: top: aqueous phase, middle: debris and proteins, bottom: chloroform.

  1. Go on to the next step quickly so the phases do not remix
  2. Pipette off the aqueous phase taking care not to suck up any of the middle or chloroform phases. Pipetting slowly helps with this.
  3. Place the aqueous phase into a new labelled eppendorf tube.
  4. Estimate the volume of the aqueous phase.
  5. Add 0.08 volumes of cold 7.5 M ammonium acetate—see attached table.
  6. Add 0.54 volumes (using the combined volume of aqueous phase and added AmAc) of cold isopropanol conversion table.
  7. Mix well by inverting.
  8. Let sit in freezer overnight. (Longer times will tend to yield more DNA, but also more contaminants.)
  9. Centrifuge for 3 min at maximum speed.
  10. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA.
  11. Add 700111 of cold 70% Ethanol and mix
  12. Centrifuge for 3 min at maximum speed.
  13. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA.
  14. Add 700111 of cold 95% Ethanol and mix
  15. Centrifuge for 3 min at maximum speed.
  16. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA. 23. Dry the pellet by letting samples stand for 1 hr or until dry.
  17. Resuspend samples with 200 ul of TE buffer. Allow to resuspend overnight in refrigerator before running a test gel using 4 gl of the DNA.

DNA Extraction Stocks

CTAB: for IL of CTAB buffer

100 ml of 1 M Tris, pH 8.0

280 ml of 5 M NaC1

40 ml of 0.5 M EDTA

20 g of CTAB (Cetyltrimethyl ammonium bromide, Amresco cat#:0833-1Kg)

TE buffer:

            [Final]                        for IL use:

            10 mM           10 ml of 1 M Tris, pH 8.0

            1 mM            2 ml of 0.5 M EDTA

1 M Tris, pH 8.0: for 1 L

121.1 g Tris (Fisher Cat#: BP152-5)

700 ml ddHO

Dissolve tris and bring to 900 ml.

pH to 8.0 with concentrated HCI (will need -50ml) Bring to 1 L.

OS M EDTA pH 8.0: for 1 L

186.12 g of EDTA (Fisher Cat#: BP120-1)

750 ml ddH20

Add about 20 g of NaOH pellets

Slowly add more NaOH until pH is 8.0, EDTA will not dissolve until the pH is near 8.0.

5 M NaC1: for 1 L

292.2 g of NaCl (Fisher Cat#: BP358-10)

700 ml ddH20

Dissolve and bring to 1 L.

References

Cullings, K.W. 1992. Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1:233-240.

Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19:11-15.

 

Genomic DNA Purification Protocol (using a DNeasy Kit)

Following CTAB extraction of high molecular weight DNA from fresh tissue, RNA digestion, protein removal and DNA shearing needs to be carried out.

  1. Add 2 1.11 RNase A (10 mg/ml, Fermentas #EN0531), incubate 550C for 10 min.
  2. Starting at step #9 of the DNeasy Plant Mini Kit (Qiagen, #69104) protocol add 67 pl AP2 Buffer to the 202111 CTAB-extracted genomic DNA (including RNase A). Incubate on ice 5 min.
  3. Continue through to end of protocol, eluting only once with 100 gl AE Buffer (allow AE to soak membrane for 5 min before centrifugation).
  4. Run 4 PI of purified genomic DNA on a agarose gel with size marker.
  5. Check DNA concentration on spectrophotometer.