Team:Harvard/color/notebook

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<li>Ran entire reaction on a gel (half in each lane), cut out appropriate band and gel purified</li>
<li>Ran entire reaction on a gel (half in each lane), cut out appropriate band and gel purified</li>
<div><a href="https://static.igem.org/mediawiki/2010/8/84/072710lut2pcrstitchgelpurification-1.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/8/84/072710lut2pcrstitchgelpurification-1.png"  width="100px"/> [click to enlarge]</a></div>
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<li>Digested gel purified LUT2, PCR purified BETA-OHASE 1, and B21 (Biobrick part in V0120 - to serve as backbone) with EcoRI and PstI to prepare for ligation of constructs into biobrick backbone</li>
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Revision as of 20:48, 28 July 2010



notebook

Week 6----07-23-2010
Week 707-26-201007-27-201007-28-201007-29-201007-30-2010


07-23-2010 [top]
  • Received primers for LUT2 and BETA-OHASE 1 - began building knockdown constructs
  • PCR reaction on RS300 to insert recognition sites. Primers listed below
  • LUT2 rxn 1: A, CP4
  • LUT2 rxn 2: CP2, CP3
  • LUT2 rxn 3: CP1, B
  • BETA-OHASE 1 rxn 1: A, CP8
  • BETA-OHASE 1 rxn 2: CP6, CP7
  • BETA-OHASE 1 rxn 3: CP5, B
  • PCR purified completed reaction
07-26-2010 [top]
  • Ran 4 ul of PCR reaction on a 1% agarose gel
  • Bands appeared to be the correct size, so started PCR stitching process
  • Ran two PCR reactions, one for each of LUT2 and BETA-OHASE 1
  • Template: Rxn 1, 2, and 3 of the appropriate construct
  • Template diluted to 1 ng/ul, used 1 ul of each template
  • Primers: A and B for both reactions
  • Size expected to be around 450 bp
  • PCR purified and ran 4 ul on a 1% agarose gel
  • PCR was not clean, decided to re-do stitching reaction with undiluted template (concentrations around 50 ng/ul, used 1 ul of each
  • Ran 4 ul on 1% agarose gel
  • Observed clear band at correct location for BETA-OHASE 1, but not for LUT2
07-27-2010 [top]
  • Redid PCR stitching for LUT2, ran 4 ul on a gel
  • Observed clear bands, including one in the right location
  • Ran entire reaction on a gel (half in each lane), cut out appropriate band and gel purified
  • Digested gel purified LUT2, PCR purified BETA-OHASE 1, and B21 (Biobrick part in V0120 - to serve as backbone) with EcoRI and PstI to prepare for ligation of constructs into biobrick backbone

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