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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. | + | 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=== | ===Practice=== | ||
| - | 1. Prepare squid tissue samples and at least 100μl of | + | 1. Prepare squid tissue samples and at least 100μl of DNA extraction buffer per tissue sample. |
| - | : | + | :DNA Extraction buffer |
::*10 mM Tris pH 8 | ::*10 mM Tris pH 8 | ||
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| - | 2. Place each tissue sample in an eppendorf tube with 100 µl | + | 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. | 3. Boil samples in waterbath for 5-10 min to inactivate Proteinase K. | ||
Revision as of 09:52, 20 July 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.
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.
