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

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)