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

From 2011.igem.org

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===Practice===
===Practice===
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1. Prepare small pieces of squid tissues
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1. Prepare squid tissue samples and at least 100μl of PCR extraction buffer per tissue sample.
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2. Add extraction buffer, 10 mm^3 squid tissue with 100 µl PCR Extraction buffer in each tube, and incubate at 50-56°C for 2-3 hrs (longer is ok). Vortex occasionally.  
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:PCR Extraction buffer
:PCR Extraction buffer
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::*200 µg/ml Proteinase K
::*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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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.
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.
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4. Spin in microfuge for 1 min, store at - 20°C.
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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.
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5. Using a pipette, transfer the supernatant (containing genomic DNA) into another tube for storage.
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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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Tip: For a 50 &mu;l PCR reaction, use 10-15 &mu;l of one of these DNA samples.
===Safety===
===Safety===

Revision as of 09:32, 20 July 2011

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Extraction of genomic DNA from squid

A method to extract genomic DNA from squid tissue.

Large sample number, very quick and dirty, adequate for PCR

protocol adapted from http://zfin.org/zf_info/zfbook/chapt9/9.3.html


Theory

How it works

Practice

1. Prepare squid tissue samples and at least 100μl of PCR extraction buffer per tissue sample.

PCR 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 PCR 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.


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