Team:Harvard/color/notebook

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<li>Gel purified LUT2 and top band from B21 (backbone)</li>
<li>Gel purified LUT2 and top band from B21 (backbone)</li>
<li>Ligated LUT2 and backbone. Transformed into TURBO e. coli and plated on LB + Amp.</li>
<li>Ligated LUT2 and backbone. Transformed into TURBO e. coli and plated on LB + Amp.</li>
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<span id="date"><a name="07-29-2010" class="date">07-29-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
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<li>Obtained colonies for LUT2. Set up four cultures in LB + Amp.</li>
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Revision as of 19:00, 29 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
  • LUT2 had the correctly sized band, but BETA-OHASE 1 had two bands, one at 400 and one at 100.
  • Checked sequences of primers, and determined that primers for BETA-OHASE 1 contained an EcoRI site
  • Ordered new primers without biobrick restriction sites for BETA-OHASE 1. Checked other primers for restriction sites, and found them to be clean.
07-28-2010 [top]
  • Gel purified LUT2 and top band from B21 (backbone)
  • Ligated LUT2 and backbone. Transformed into TURBO e. coli and plated on LB + Amp.
07-29-2010 [top]
  • Obtained colonies for LUT2. Set up four cultures in LB + Amp.

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