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

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==Extraction of Genomic DNA from Squid==
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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).'''
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==Extraction of genomic DNA from squid==
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===Theory===
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A method to extract genomic DNA from squid tissue.
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A tissue sample is incubated with a small quantity of DNA Extraction Buffer, containing the following compounds:
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Large sample number, very quick and dirty, adequate for PCR
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:'''EDTA''': A chelating agent. inactivates metal-cofactor enzymes that might damage DNA.
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:'''Proteinase K''': A broad spectrum proteinase which rapidly inactivates RNAases and DNAases to prevent degradation of DNA.
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:'''Tris pH 8''': Provides physiological pH.
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:'''Triton X-100''': A non-ionic surfactant useful for lysing cells.
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protocol adapted from http://zfin.org/zf_info/zfbook/chapt9/9.3.html
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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.
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===Theory===
 
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How it works
 
===Practice===
===Practice===
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Protocol adapted from [http://zfin.org/zf_info/zfbook/chapt9/9.3.html here].
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====Materials====
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:'''DNA Extraction buffer'''
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::*10 mM Tris pH 8
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::*2 mM EDTA
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a
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::*0.2% Triton X-100
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::*200 µg/ml Proteinase K
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:'''squid tissue sample'''
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::*~1 mm<sup>3</sup> cut into several pieces
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'''Optional:''' instead of cubes of squid tissue, crush a tissue sample under liquid nitrogen with a pestle and mortar.
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====Method 1====
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This protocol is most suitable for samples consisting of 1-20, diploid, 2-3 day old embryos. Embryos need not be removed from their chorions. DNA prepared with this procedure is only good for PCR analysis, but is unsuitable for digests and Southern blots. Because PCR does not require high-molecular-weight DNA, samples can be vortexed and frozen.
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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.
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1. Transfer embryo(s) into microfuge tube and remove excess liquid with drawn-out Pasteur pipette.
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2. Boil samples in waterbath for 5-10 min to inactivate Proteinase K.
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2. Add extraction buffer, 50 µl or 10 µl per embryo whichever is larger, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally.  
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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.
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4. Pipette the supernatant (containing genomic DNA) into another tube for storage.
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PCR Extraction buffer
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'''Tip''': For a 50 &mu;l PCR reaction, use 10-15 &mu;l of one of these DNA samples.
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10 mM Tris pH 8
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====Method 2====
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2 mM EDTA
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0.2% Triton X-100
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200 µg/ml Proteinase K
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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.
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3. Boil samples in waterbath for 5-10 min to inactivate Proteinase K.
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2. Add 200µl EtOH,mix, and place on ice for 20-30 min.
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4. Spin in microfuge for 1 min, store at - 20°C.
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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.
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5. Use 10-15 µl to set up PCR reaction in a total volume of 50 µl, proceed with your favorite PCR protocol.
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4. Resuspend the DNA in 40µl TE, store at -20°C.
===Safety===
===Safety===
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The safety implication of the procedure.
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*Transport of liquid nitrogen: consult your supplier and/or department health and safety officer about safe transfer of liquid nitrogen.
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*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.
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*'''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.
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*'''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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Latest revision as of 20:27, 21 September 2011

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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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