Team:Cambridge/Protocols/Filter Paper

From 2011.igem.org

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==Extraction of DNA from Filter Paper==
==Extraction of DNA from Filter Paper==
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A method of purifying DNA, usually plasmid DNA, from a blotting paper.  
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A method of purifying DNA, usually plasmid DNA, from blotting paper.  
===Theory===
===Theory===
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Spotting DNA on a 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, if contamination is avoided.
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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, if contamination is avoided.
===Practice===
===Practice===
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'''''DNA spotting on filter paper'''''
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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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:# Spot 2 μL of DNA solution on a 2 cm square piece of clean filter paper.
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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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:#*'''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 of plasmid.  
:# Leave spots to dry at room temperature for 1 hour.
:# Leave spots to dry at room temperature for 1 hour.
:#*'''Make sure the spots are protected from contamination by dust, dirt and aerosols.
:#*'''Make sure the spots are protected from contamination by dust, dirt and aerosols.
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'''''Extraction of DNA'''''
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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 μl [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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:# Cut out a portion of the plasmid-containing filter paper (half of the blot), and apply 50 μl of [http://www.qiagen.com/faq/faqview.aspx?faqid=199&page=4&showall=1&faqcategoryid=0&menuitemid=56 QIAGEN elution buffer] in an Eppendorf tube.
:# Spin in the microcentrifuge for 1 min to elute the DNA from the paper.
:# Spin in the microcentrifuge for 1 min to elute the DNA from the paper.
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'''''Transformation of E.coli with purified DNA'''''
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:'''''Transformation of E.coli with purified DNA'''''
:# Add 250 μl 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 μl of the plasmid DNA.  
:# Add 250 μl 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 μl of the plasmid DNA.  
:#* '''When pipetting the plasmid, use the pipette tip to squeeze some fluid off the filter paper if necessary.'''
:#* '''When pipetting the plasmid, use the pipette tip to squeeze some fluid off the filter paper if necessary.'''
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The procedure does not involve any safety considerations.
The procedure does not involve any safety considerations.
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{{Template:Team:Cambridge/CAM_2011_PROTOCOL_FOOT}}

Latest revision as of 20:29, 21 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, if contamination is avoided.

Practice

DNA spotting on filter paper
  1. Spot 2 μL of DNA solution on a 2 cm 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 of 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 of QIAGEN elution 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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