Team:Cambridge/Protocols/Filter Paper

From 2011.igem.org

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==Protocol Name==
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==Extraction of DNA from Filter Paper==
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protocol description
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A method of purifying DNA, usually plasmid DNA, from blotting paper.
===Theory===
===Theory===
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How it works
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Spotting DNA on filter paper is an easy and safe way of plasmid shipment widely used in the scientific world. Negatively charged DNA is firmly bound to cellulose fibers of the blotting paper, and remains stable at room temperature for several days when contamination is avoided. 
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===Practice===
===Practice===
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How to do it in the lab
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'''''DNA spotting on filter paper'''''
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:# Spot 2 μL of DNA solution on a 2cm square piece of clean filter paper.
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{Standard layout for procedures is to use: 
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:#*'''The DNA sample should be at a concentration of at least 100 ng/μl. The solution should contain 1 μl of 10 mM Tris, pH 7.6, per 500 ng per plasmid.
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:# Leave spots to dry at room temperature for 1 hour.
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*'''''<Procedure title - aka what you are doing>'''''
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:#*'''Make sure the spots are protected from contamination by dust, dirt and aerosols.
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:# <step 1>
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:# <step 2>
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:#* '''<additional notes/important information regarding the previous step>'''
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the text within the < > is what should be written, don't include < > in actual writeup :P
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if in doubt see the gel electrophoresis protocol
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*'''''Extraction of DNA'''''
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:# Cut out a portion of the plasmid-containing filter paper (half of the blot), and apply 50 &mu;l [http://www.qiagen.com/faq/faqview.aspx?faqid=199&page=4&showall=1&faqcategoryid=0&menuitemid=56 QIAGEN elusion buffer] in an Eppendorf tube.
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:# Spin in the microcentrifuge for 1 min to elute the DNA from the paper.
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}
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:'''''Transformation of E.coli with purified DNA'''''
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:# Add 250 &mu;l of SOC medium to a 1.5 ml Eppendorf tube of OD600 competent cells and [https://2011.igem.org/Team:Cambridge/Protocols/Transformation_of_E.Coli  transform] with 1 &mu;l of the plasmid DNA.
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:#* '''When pipetting the plasmid, use the pipette tip to squeeze some fluid off the filter paper if necessary.'''
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:#* '''Store the remaining solution together with the filter paper in a freezer.'''
===Safety===
===Safety===
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The safety implication of the procedure.
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The procedure does not involve any safety considerations.
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{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Revision as of 14:42, 19 September 2011

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Extraction of DNA from Filter Paper

A method of purifying DNA, usually plasmid DNA, from blotting paper.

Theory

Spotting DNA on filter paper is an easy and safe way of plasmid shipment widely used in the scientific world. Negatively charged DNA is firmly bound to cellulose fibers of the blotting paper, and remains stable at room temperature for several days when contamination is avoided.

Practice

DNA spotting on filter paper

  1. Spot 2 μL of DNA solution on a 2cm square piece of clean filter paper.
    • The DNA sample should be at a concentration of at least 100 ng/μl. The solution should contain 1 μl of 10 mM Tris, pH 7.6, per 500 ng per plasmid.
  2. Leave spots to dry at room temperature for 1 hour.
    • Make sure the spots are protected from contamination by dust, dirt and aerosols.
  • Extraction of DNA
  1. Cut out a portion of the plasmid-containing filter paper (half of the blot), and apply 50 μl QIAGEN elusion buffer in an Eppendorf tube.
  2. Spin in the microcentrifuge for 1 min to elute the DNA from the paper.
Transformation of E.coli with purified DNA
  1. Add 250 μl of SOC medium to a 1.5 ml Eppendorf tube of OD600 competent cells and transform with 1 μl of the plasmid DNA.
    • When pipetting the plasmid, use the pipette tip to squeeze some fluid off the filter paper if necessary.
    • Store the remaining solution together with the filter paper in a freezer.

Safety

The procedure does not involve any safety considerations.


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