Team:Copenhagen/Project/Experimental

From 2011.igem.org

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<li>You perform a Miniprep on your culture to purify your amplified plasmid </li>
<li>You perform a Miniprep on your culture to purify your amplified plasmid </li>
<li>Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site</li>
<li>Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site</li>
-
<li>Run the digest of the plasmid and hope to see a single band and not two (in the latter case your mutations failed) </li>
+
<li>Run the digest of the plasmid and hope to see a number of bands corresponding to the number of restriction sites in the plasmid backbone. In the latter case your mutations failed </li>
<li>Add prefix and suffix containing primers for the CYP in question and amplify with PCR</li>
<li>Add prefix and suffix containing primers for the CYP in question and amplify with PCR</li>
-
<li>Purify with DNA purification kit</li>
+
<li>Purify with DNA Purification kit</li>
<li>Cut with restrictionsenzymes EcoR1 and Pst1</li>
<li>Cut with restrictionsenzymes EcoR1 and Pst1</li>
<li>Run on a gel and cut the wanted band out </li>
<li>Run on a gel and cut the wanted band out </li>
-
<li>Put it in the freezer</li>
+
<li>Extract the DNA from the gel pieces by using a Gel Extraction kit</li>
<li>Cut the linarized plasmid backbone with Pst1 and EcoR1 </li>
<li>Cut the linarized plasmid backbone with Pst1 and EcoR1 </li>
<li>Ligate CYP and Plasmid </li>
<li>Ligate CYP and Plasmid </li>

Revision as of 15:16, 11 July 2011


How to
Mutate

  • Analysis have made you realise the need for mutations in the CYP in order to remove resctrictionsites
  • You design primers that fits your template but contains the altered base
  • You mix your primers with the template, dNTP, polymerase and HF buffer and run a non amplifying PCR
  • Digest the parental template DNA (which is methylated) with Dpn1 (which cut methylated DNA only)
  • Transform XL1-Blue competent cells with the mutated plasmid
How to
make a BioBrick

  • Once you have a colony on your plate you take it out and place it in and overnight culture
  • You perform a Miniprep on your culture to purify your amplified plasmid
  • Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site
  • Run the digest of the plasmid and hope to see a number of bands corresponding to the number of restriction sites in the plasmid backbone. In the latter case your mutations failed
  • Add prefix and suffix containing primers for the CYP in question and amplify with PCR
  • Purify with DNA Purification kit
  • Cut with restrictionsenzymes EcoR1 and Pst1
  • Run on a gel and cut the wanted band out
  • Extract the DNA from the gel pieces by using a Gel Extraction kit
  • Cut the linarized plasmid backbone with Pst1 and EcoR1
  • Ligate CYP and Plasmid
  • Transform in XL1-Blue
  • Overnight culture
  • Hurra - You have a Biobrick
How to
make a CyperMan

  • Cut the newly made Cyp BioBrick with restrictionsenzymes Xba and Pst1
  • Run on a gel and cut the wanted band out
  • Put it in the freezer
  • Cut the purified BioBrick J004500 ekspression vector from the kit with Spe1 and EcoR1
  • Ligate CYP and Vector
  • Transform in XL1-Blue
  • Overnight culture
  • Hurra - You have a CyperMan


Comments or questions to the team? Please mail us at igemcopenhagen@gmail.com

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