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Team:Cambridge/Protocols/Extraction of genomic DNA from squid
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==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. | + | A method to extract genomic DNA from squid tissue. The method is quick and provides a large, dirty sample; adequate for PCR. '''In fact, we were unable to clone from squid DNA obtained through this protocol. Such difficulties manipulating cephalopod DNA are not new and are believed to be due to protein contaminants (personal correspondence with Wendy Crookes-Goodson).''' |
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===Theory=== | ===Theory=== | ||
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===Practice=== | ===Practice=== | ||
| + | Protocol adapted from [http://zfin.org/zf_info/zfbook/chapt9/9.3.html here]. | ||
1. Prepare squid tissue samples and at least 100μl of DNA extraction buffer per tissue sample. | 1. Prepare squid tissue samples and at least 100μl of DNA extraction buffer per tissue sample. | ||
Revision as of 11:18, 14 September 2011
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. In fact, we were unable to clone from squid DNA obtained through this protocol. Such difficulties manipulating cephalopod DNA are not new and are believed to be due to protein contaminants (personal correspondence with Wendy Crookes-Goodson).
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
Protocol adapted from [http://zfin.org/zf_info/zfbook/chapt9/9.3.html here].
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
Advice for all reagents is the same: in case of contact with skin or eyes rinse thoroughly and consult a physician. There is a rick of serious eye injury in case of contact with eyes.
All materials coming into contact with squid tissue must be autoclaved. Scalpels or razorblades used in dissection should be disposed off in a designated sharps bin.
