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Team:Cambridge/Protocols/Extraction of cleaner genomic DNA from squid
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==Extraction of cleaner genomic DNA from squid== | ==Extraction of cleaner genomic DNA from squid== | ||
| - | As the quick and dirty method to extract genomic DNA failed to produce genomic DNA suitable for PCR, two new protocols are carried out as an attempt to obtain cleaner genomic DNA. This protocol produces | + | As the quick and dirty method to extract genomic DNA failed to produce genomic DNA suitable for PCR, two new protocols are carried out as an attempt to obtain cleaner genomic DNA. This protocol produces cleaner preparations and probably somewhat higher yields of genomic DNA. This is adapted from an improved version of the protocol for Zebrafish. |
===Theory=== | ===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. Incubate at 50 for about an 3 hr. Mix occasionally | |
| + | |||
| + | :DNA Extraction Buffer | ||
| + | ::*10mM Tris pH 8.2 | ||
| + | ::*10mM EDTA | ||
| + | ::* 200 mM NaCl | ||
| + | ::*0.5% SDS | ||
| + | ::*200 µg/ml proteinase K | ||
| + | |||
| + | 2. Add 200µl EtOH,mix, and place on ice for 20-30 min. | ||
| + | |||
| + | 3. Centrifuge in microfuge for 10 min, remove supernatant and add 400 µl 70% EtOH. Spin again for 2 min, remove liquid and dry pellet. | ||
| + | |||
| + | 4. Resuspend the DNA in 40µl TE, store at -20. | ||
| + | µl Proceed with PCR reaction. | ||
| - | |||
===Safety=== | ===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. | ||
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | ||
Latest revision as of 10:52, 16 August 2011
Extraction of cleaner genomic DNA from squid
As the quick and dirty method to extract genomic DNA failed to produce genomic DNA suitable for PCR, two new protocols are carried out as an attempt to obtain cleaner genomic DNA. This protocol produces cleaner preparations and probably somewhat higher yields of genomic DNA. This is adapted from an improved version of the protocol for Zebrafish.
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. Incubate at 50 for about an 3 hr. Mix occasionally
- DNA Extraction Buffer
- 10mM Tris pH 8.2
- 10mM EDTA
- 200 mM NaCl
- 0.5% SDS
- 200 µg/ml proteinase K
2. Add 200µl EtOH,mix, and place on ice for 20-30 min.
3. Centrifuge in microfuge for 10 min, remove supernatant and add 400 µl 70% EtOH. Spin again for 2 min, remove liquid and dry pellet.
4. Resuspend the DNA in 40µl TE, store at -20. µl Proceed with PCR reaction.
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.
