Team:Cambridge/Protocols/Extraction of genomic DNA from squid

Extraction of genomic DNA from squid

A method to extract genomic DNA from squid tissue. The method is quick and provides a large, dirty sample; adequate for PCR.

Protocol adapted from [http://zfin.org/zf_info/zfbook/chapt9/9.3.html here].

Theory

A tissue sample is incubated with a small quantity of DNA Extraction Buffer, containing the following compounds:

EDTA: A chelating agent. Inactivated metal-activated enzymes that might damage DNA.

Proteinase K: A broad spectrum proteinase which rapidly inactivates RNAases and DNAases to prevent degradation of DNA.

Tris pH 8: Provides physiological pH.

Triton X-100: A non-ionic surfactant useful for lysing cells.

After incubation the mixture is centrifuged to pellet cell fragments and remaining tissue. The DNA sample is quite dirty: adequate for PCR but for a pure sample further processing is necessary.

Practice

1. Prepare squid tissue samples and at least 100μl of DNA extraction buffer per tissue sample.

DNA Extraction buffer

• 10 mM Tris pH 8
• 2 mM EDTA
• 0.2% Triton X-100
• 200 µg/ml Proteinase K

squid tissue sample

• ~1 mm³ cut into several pieces

2. Place each tissue sample in an eppendorf tube with 100 µl DNA Extraction buffer, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally.

3. Boil samples in waterbath for 5-10 min to inactivate Proteinase K.

4. Spin in microfuge at 13000 rpm for 5 minutes. The supernatant should contain the DNA while the proteins and cell fragments are in the pellet.

5. Using a pipette, transfer the supernatant (containing genomic DNA) into another tube for storage.

Tip: For a 50 μl PCR reaction, use 10-15 μl of one of these DNA samples.

Safety

The safety implication of the procedure.