Team:Harvard/color/notebook

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notebook

Week 6----07-23-2010
Week 707-26-201007-27-201007-28-201007-29-201007-30-2010
Week 8--08-04-201008-05-201008-06-2010


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.
08-04-2010 [top]
  • Received new primers for BETA-OHASE 1 - began building knockdown constructs for that and LYC
  • PCR reaction on RS300 to insert recognition sites. Primers listed below
  • LYC rxn 1: A, CP12
  • LYC rxn 2: CP10, CP11
  • LYC rxn 3: CP9, B
  • BETA-OHASE 1 rxn 1: A, CP16
  • BETA-OHASE 1 rxn 2: CP14, CP15
  • BETA-OHASE 1 rxn 3: CP13, B
08-05-2010 [top]
PCR Stitching of LYC and BETA-OHASE 1 amiRNA parts; Digestion and Ligation into biobrick backbone
  • Ran PCR reaction on a 1% agarose gel.
  • Initial PCR was successful. Gel purified parts
  • Performed PCR Stitching for each of BETA-OHASE 1 and LYC
  • Template: Rxn 1, 2, and 3 of the appropriate construct
  • Used 1 ul of each template - concentrations were all around 25 ng/ul
  • 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 successful. Gel purified products
  • Digested products with EcoRI and PstI to ligate into V0120 backbone.
  • Successful digestion - gel purified and ligated into backbone digested EcoRI/PstI last week
  • Transformed into TURBO E. coli and plated on LB + Amp
PCR Amplification of petal promoters
  • Ran PCR to amplify petal promoters
  • Template: Arabidopsis genomic DNA, 80ng
  • Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)
  • Protocol: Phusion
  • Ran completed reaction on 1% agarose gel
  • PCR Failed. Retry with PFx
  • Template: Arabidopsis genomic DNA, 80ng
  • Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)
  • Protocol: PFx, extension time 2 min
  • Ran completed reaction on 1% agarose gel
08-06-2010 [top]
  • Got colonies for C3 (LYC) but not for C2 (BETA-OHASE 1). Picked four colonies and set up cultures for C3
  • Redid digestion on C2, and digested PCR products C4-6 with EcoRI and PstI. Also digested B21 with EcoRI and PstI with TSAP to obtain backbone.
  • C2 and B21 digests worked, C4 had a PstI site inside the part and C5/C6 had EcoRI sites
  • Gel purified C2 and B21 (top) bands
  • Redigested C4-C6 with XbaI and SpeI, as well as B21 with XbaI and SpeI with TSAP to obtain backbone
  • Gel ran strangely - ladder didn't show. Cut out bands and gel purified, then ran second gel to check sizes again
  • Miniprepped cultures set up in the morning, and sent out for sequencing

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