Extracting DNA from
DNA Extraction Protocol
This protocol produces high quality DNA that can be stored for at least 6 years at -15 °C. The starting point for the extraction is assumed to be a piece of tissue preserved in ethanol or DMSO+NaCl (see preserving DNA).
1. Pellet tissue out of the storage solution by centrifugation for few minutes at 14,000 rpm.
2. Transfer a one-to-two-Drosophila-sized piece of tissue into a sterile 1.7ml microfuge tube.
3. Add 600 ml CTAB. Add 6 ml Proteinase K (20 mg ml-1). Add 60 ml 5M NaCl. Mix.
4. Digest at 42 °C overnight or 55 °C for few hours.
1. Centrifuge digested samples for 5 minutes at 12,000 rcf.
2. Pipette 600 ml of supernatant into a sterile 1.7ml microfuge tube. Add 600 ml CI (24:1) [chloroform:isoamyl-alcohol], invert gently several times, centrifuge for 10 minutes at 12,000 rcf.
3. Pipette supernatant into a sterile 1.7ml microfuge tube. Add equal volume of PCI (25:24:1), invert gently several times, centrifuge for 10 minutes at 12,000 rcf.
4. Repeat step 3 until the interface between organic and aqueous phases is clean.
5. Pipette supernatant into a sterile 1.7ml microfuge tube. Add equal volume of CI, invert gently several times, centrifuge for 5 minutes at 12,000 rcf.
6. Pipette supernatant into a sterile 1.7ml microfuge tube. Add 1/10th vol 3M NaOAc. Add 2 vols of Ð20 °C 100% EtOH. Invert several times. Incubate at -20 °C for one hour.
7. Centrifuge for 30 minutes at 12,000 rcf. Decant off the supernatant.
8. Wash the pellet in 75% ethanol. Pipette off the liquid. Repeat.
9. Dry the pellet at 37 °C for approximately 30-40 mins, or on the bench-top overnight.
10. Dissolve the pellet in 50ml of 10mM Tris-HCl pH8.3.
1. Make 1.4% agarose minigel in 0.5x TBE containing ethidium bromide (EtBr, 10 ul * 0.5 mg/ml per 25 ml gel).
2. Load ~6ul sample and appropriate volume of mass ladder and electrophorese at 136V for 25 mins.
3. Visualize gel on UV transilluminator and photograph.
Qiagen's DNeasy kits also can be used successfully to purify scyphozoan DNA, although the results are less consistent than those using the CTAB-Phenol/Chloroform protocol.
GelStar is a suitable alternative to ethidium bromide for vizualizing DNA.
by M. N Dawson