User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/21

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Cellular Adhesion

  • Review of all standard protocols used for this project
    • PCR
      • Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of PCR product (or .4ul of N. gonorrhea DNA or .4ul PCR purification)
      • Protocol:
        • 5mins@95C
        • 20secs@94C
        • 20secs@55C
        • 1min@68C
        • REPEAT 2-4, 34 times
        • 5mins@70C
        • FOREVER@4C
    • PCR Purification
      • Used MinElute columns
      • Eluted in 10ul of water
    • Gel
      • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
    • Digest
      • Template for IGA (single): 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
      • Template for IGA (pairs): 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
      • Template for tags: 3ul DNA (each), 2ul NEB2, .5ul EcoR1, .5ul Pst1, rest water (20ul rxn)
      • Thermocycler protocol: 1hr@37C, 20mins@80C
    • Construct Digest
      • Beginning to assemble parts into a functional construct
      • Template: 200ng DNA, 2ul NEB2, .2ul BSA, .5ul of each enzyme, rest water (20ul)
      • The first part should be cut with EcoR1 and Spe1
      • The second part should be cut with Xba1 and Pst1
    • Ligation
      • 6ul of IGA digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
      • Template for IGA (assemble): .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
      • Protocol: 30mins@roomtemp,10mins@65C
    • Transformation
      • 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
      • 45secs heat shock at 42C using water bath
      • Added 300ul of SOC
      • Incubated for 1hr at 37C
      • Plated on Amp/Cl and grew up at 37C overnight
    • Liquid Culture
      • Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from plate colonies
      • Grew up in 37C overnight
    • Glycerol
      • Took 1.6ml of liquid culture and created 10% glycerol stocks
    • Miniprep
      • Extracted DNA from remaining liquid culture (~6ml)
      • Eluted in 50ul water
    • Sequencing
      • Template: 3ul Miniprep DNA, .3ul primer, 6.7ul water
      • Template: 1ul PCR DNA, .3ul primer, 8.7ul water
    • Colony PCR
      • Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
      • Picked three colonies from each plated sample to PCR


  • Status
    • IGA
      • beta-core: incomplete
      • signal sequence: complete (made on 8/5/2007, in freezer box position 1.3)
    • Leucine Zippers
      • GCN4 Leucine Zipper: incomplete (due to bad primers)
      • Fos Zipper: complete (made on 8/6/2007, in freezer box position 1.4)
      • JunB Zipper: complete (made on 8/6/2007, in freezer box position 1.5)
    • Tags
      • 6His Tag: incomplete
      • FLAG Tag: incomplete
      • Myc Tag: incomplete
      • HA Tag: incomplete


  • Plan for tomorrow
    • Start from scratch with tag primers (3 of each) -> get all the way to cultures
    • Same idea for GCN4 zipper
    • Use new primers (with extended cutting region) to start IGAbc pieces -> try to assemble B+C and C+D components
    • Clean out -80/-20/4 of old materials (unlabeled or useless)
    • Record 8/20/2007 sequence run results


  • Rest of the week
    • Sequencing submitted goals:
      • Tags (Wednesday)
      • Zipper (Wednesday)
      • IGAbc (Friday)
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