Team:Cambridge/Experiments/Synthetic Reflectin PCR and Construction of GA1 to 6

From 2011.igem.org

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===Practice===
===Practice===
====Amplification====
====Amplification====
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*Cut out a portion of the plasmid-containing filter paper, and apply some elusion buffer (QIAGEN) 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 elusion buffer (QIAGEN) in an eppendorf tube.
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*Spin in the micro centrifuge for 1 min. This should elute the DNA from the paper into solution; paper residue can now be discarded.
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*Spin in the micro centrifuge for 1 min to elute the DNA from the paper.
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*[https://2011.igem.org/Team:Cambridge/Protocols/Transformation_of_E.Coli  Transform] competent E.coli cells using the solution containing the plasmids.
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*Add 250 μl of SOC 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. At this point, the eluted DNA ('''with''' paper) was then stored in the freezer.
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*Incubate the bacteria overnight under selection on LB agar. Colonies should have grown by the following morning.
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*Plate out 10 μl and 100 μl on LB agar plates
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====Miniprep extraction====
====Miniprep extraction====
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Healthy colonies of transformed E. coli were observed after incubation in LB agar, indicating the success of the amplification procedure.
Healthy colonies of transformed E. coli were observed after incubation in LB agar, indicating the success of the amplification procedure.
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'''storage, quantity of elusion buffer, incubation time, selection antibiotic, check with Nanodrop, check miniprep paper dissolved?''
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'''selection antibiotic, check with Nanodrop, check miniprep paper dissolved?''
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{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Revision as of 08:53, 3 August 2011

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Contents

Amplification of Synthesised Reflectin Genes

The sample plasmids containing the reflectin gene are first extracted from filter paper, and then used in the transformation of competent E. coli cells. A standard miniprep can then be used to recover the plasmid DNA from the bacteria. The purpose of this procedure is to amplify and store the plasmids' DNA.

Practice

Amplification

  • Cut out a portion of the plasmid-containing filter paper (half of the blot), and apply 50 μl elusion buffer (QIAGEN) in an eppendorf tube.
  • Spin in the micro centrifuge for 1 min to elute the DNA from the paper.
  • Add 250 μl of SOC 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. At this point, the eluted DNA (with paper) was then stored in the freezer.
  • Plate out 10 μl and 100 μl on LB agar plates


Miniprep extraction

  • Transfer some bacteria from the colonies to liquid culture, and incubate for a further 16 hours.
  • Perform a standard miniprep.

Results

Healthy colonies of transformed E. coli were observed after incubation in LB agar, indicating the success of the amplification procedure.

'selection antibiotic, check with Nanodrop, check miniprep paper dissolved?



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