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

From 2011.igem.org

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(Practice)
(Extraction of genomic DNA from squid)
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==Extraction of genomic DNA from squid==
==Extraction of genomic DNA from squid==
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A method to extract genomic DNA from squid tissue.
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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.
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Large sample number, very quick and dirty, adequate for PCR
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Protocol adapted from [http://zfin.org/zf_info/zfbook/chapt9/9.3.html here].
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protocol adapted from http://zfin.org/zf_info/zfbook/chapt9/9.3.html
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===Theory===
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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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:'''EDTA''': A chelating agent. Inactivated metal-activated 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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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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1. Prepare squid tissue samples and at least 100μl of PCR extraction buffer per tissue sample.
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1. Prepare squid tissue samples and at least 100μl of DNA extraction buffer per tissue sample.
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:PCR Extraction buffer
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: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 PCR Extraction buffer, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally.
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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

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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.

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.