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Team:Cambridge/Protocols/Extraction of genomic DNA from squid
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| - | {{Template:Team:Cambridge/ | + | {{Template:Team:Cambridge/CAM_2011_PROTOCOL_HEAD}} |
| - | + | ==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 known and are believed to be due to protein contaminants (personal correspondence with Wendy Crookes-Goodson).''' | |
| - | ==Extraction of | + | |
| - | A method to extract genomic DNA from squid tissue. The method is quick and provides a large, dirty sample; adequate for PCR. | + | |
| - | + | ||
| - | + | ||
===Theory=== | ===Theory=== | ||
A tissue sample is incubated with a small quantity of DNA Extraction Buffer, containing the following compounds: | A tissue sample is incubated with a small quantity of DNA Extraction Buffer, containing the following compounds: | ||
| - | :'''EDTA''': A chelating agent. | + | :'''EDTA''': A chelating agent. inactivates metal-cofactor enzymes that might damage DNA. |
:'''Proteinase K''': A broad spectrum proteinase which rapidly inactivates RNAases and DNAases to prevent degradation of DNA. | :'''Proteinase K''': A broad spectrum proteinase which rapidly inactivates RNAases and DNAases to prevent degradation of DNA. | ||
:'''Tris pH 8''': Provides physiological pH. | :'''Tris pH 8''': Provides physiological pH. | ||
| Line 18: | Line 14: | ||
===Practice=== | ===Practice=== | ||
| + | Protocol adapted from [http://zfin.org/zf_info/zfbook/chapt9/9.3.html here]. | ||
| - | + | ====Materials==== | |
| - | + | :'''DNA Extraction buffer''' | |
| - | :DNA Extraction buffer | + | |
| - | + | ||
::*10 mM Tris pH 8 | ::*10 mM Tris pH 8 | ||
::*2 mM EDTA | ::*2 mM EDTA | ||
| + | a | ||
::*0.2% Triton X-100 | ::*0.2% Triton X-100 | ||
::*200 µg/ml Proteinase K | ::*200 µg/ml Proteinase K | ||
| - | :squid tissue sample | + | :'''squid tissue sample''' |
::*~1 mm<sup>3</sup> cut into several pieces | ::*~1 mm<sup>3</sup> cut into several pieces | ||
| + | '''Optional:''' instead of cubes of squid tissue, crush a tissue sample under liquid nitrogen with a pestle and mortar. | ||
| + | |||
| + | ====Method 1==== | ||
| + | |||
| + | 1. Place each tissue sample in a microcentrifuge tube with 100 µl DNA Extraction buffer, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally. | ||
| + | |||
| + | 2. Boil samples in waterbath for 5-10 min to inactivate Proteinase K. | ||
| + | |||
| + | 3. 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. | ||
| + | |||
| + | 4. Pipette 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. | ||
| - | 2 | + | ====Method 2==== |
| - | + | 1. Place each tissue sample in a microcentrifuge tube with 100 µl DNA Extraction buffer, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally. | |
| - | + | 2. Add 200µl EtOH,mix, and place on ice for 20-30 min. | |
| - | + | 3. Centrifuge in microcentrifuge 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°C. | |
===Safety=== | ===Safety=== | ||
| - | + | *Transport of liquid nitrogen: consult your supplier and/or department health and safety officer about safe transfer of liquid nitrogen. | |
| + | *Handling of liquid nitrogen: Reduce contact with skin to avoid cold burns. Do not accompany liquid nitrogen in any enclosed space and work in a well-ventilated area, or else any spillage or leakage may create a risk of asphyxiation. Liquid nitrogen boils off very quickly, so do not put liquid nitrogen in closed vessels that cannot withstand the pressure. | ||
| + | *'''DNA extraction buffer:''' Advice for all components is the same: in case of contact with skin or eyes rinse thoroughly and consult a physician. There is a risk of serious eye injury in case of contact with eyes. | ||
| + | *'''Squid tissue:''' 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/ | + | {{Template:Team:Cambridge/CAM_2011_PROTOCOL_FOOT}} |
Latest revision as of 20:27, 21 September 2011
Contents |
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 known 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. inactivates metal-cofactor 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].
Materials
- DNA Extraction buffer
- 10 mM Tris pH 8
- 2 mM EDTA
a
- 0.2% Triton X-100
- 200 µg/ml Proteinase K
- squid tissue sample
- ~1 mm3 cut into several pieces
Optional: instead of cubes of squid tissue, crush a tissue sample under liquid nitrogen with a pestle and mortar.
Method 1
1. Place each tissue sample in a microcentrifuge tube with 100 µl DNA Extraction buffer, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally.
2. Boil samples in waterbath for 5-10 min to inactivate Proteinase K.
3. 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.
4. Pipette 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.
Method 2
1. Place each tissue sample in a microcentrifuge tube with 100 µl DNA Extraction buffer, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally.
2. Add 200µl EtOH,mix, and place on ice for 20-30 min.
3. Centrifuge in microcentrifuge 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°C.
Safety
- Transport of liquid nitrogen: consult your supplier and/or department health and safety officer about safe transfer of liquid nitrogen.
- Handling of liquid nitrogen: Reduce contact with skin to avoid cold burns. Do not accompany liquid nitrogen in any enclosed space and work in a well-ventilated area, or else any spillage or leakage may create a risk of asphyxiation. Liquid nitrogen boils off very quickly, so do not put liquid nitrogen in closed vessels that cannot withstand the pressure.
- DNA extraction buffer: Advice for all components is the same: in case of contact with skin or eyes rinse thoroughly and consult a physician. There is a risk of serious eye injury in case of contact with eyes.
- Squid tissue: 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.
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