Genomic DNA Isolation

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Isolation of Plant Genomic DNA for GISH

1.     Grind 5 g plant leaves in liquid nitrogen.  Never let the plant material thaw.  Pour powder into a 250 ml flask.

2.     Suspend powdered material in 15 ml of 2X CTAB buffer and incubate in a water bath at 65°C for 1 hr at slow shaking.

3.     Let the flasks cool down to room temperature.  Add 15 ml chloroform/isoamyl alcohol (24:1) and swirl the flask until it makes an emulsion (place the flask on a rotary shaker 15 min -1 hr at room temperature).

4.     Pour sample into a centrifuge tube and spin at 10000 rpm for 15 min at room temperature.

5.     Transfer the supernatant to a clean tube and add 2/3 volumes of isopropyl alcohol.  Invert the tube several times.  Hook out the precipitated DNA and transfer it to a clean 10 ml tube.

6.     Wash the DNA pellet twice with 70% ethanol.  Pour off the ethanol and let the pellet air dry for 1 hr.

7.     Add 500 µl of TE and transfer the DNA into a 1.5 ml tube.  Add 5 µl RNase A (10 mg/ml) and leave at 4°C overnight or 37°C for 1 hr.

8.     Add 500 µl phenol, mix well, spin for 5 min.  Transfer the supernatant to another tube.

9.     Add 250 µl each of phenol and chloroform, mix well, spin for 5 min.  Transfer the supernatant to another tube.

10.   Add 500 µl chloroform, mix well, spin 5 min.  Transfer the supernatant to a 10 ml tube.

11.   Add TE to 3 ml then add 1/10 volume of 3M NaAc and 2 volumes of 100% ethanol and invert the tube several times.

12.   Wash the precipitated DNA twice with 70% ethanol.  Transfer the DNA to a 1.5 ml tube.  Dry the DNA pellet and dissolve into 500 µl TE.

13.   Determine the DNA concentration by running a mini-gel.

Notes:

a.     2X CTAB buffer: 1.4 M Nacl; 100 mM Tris ph 8.0; 2% CTAB (Hexadecyltrimethylamonium bromide); 20 mM EDTA; 0.5% Na Bisulfite; 1% 2-mercaptoethanol (2-me).

b.     When making the CTAB buffer it is fastest to add the NaCl after the other ingredients (except the 2-me) are in solution.  Do not add the 2-me until just before use.  The solution without the 2-me can be stored at room temperature.

c.     For genomic in situ hybridization (GISH) purpose, the cleaner the genomic DNA, the better the biotin-labelled probe.  Therefore, the isolated DNA may be further purified by CsCl purification procedures.  However, the quality of DNA isolated with above protocol is good enough for GISH in our hands.