User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/04

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  • PCR Purification
    • Used MinElute columns to purify B, C, D, and IgAbc complete (no muts) PCR product from yesterday
    • Eluted in 10ul of 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 (Signal Sequence, IgAbc R, Fos, JunB, GCN4)
    • Plated picked colonies on Amp/Cl plate and grew up at 37C all day and overnight
    • Protocol:
      • 95C for 15 mins
      • 94C for 30 secs
      • 55C for 30 secs
      • 68C for 1 min
      • REPEAT 2-4 39 times
      • 68C for 10 mins
      • 4C FOREVER
IgAbc Part 1(B), IgAbc Part 2(C), IgAbc Part 3(D), log-2 ladder, IgAbc complete (D, no muts)
  • Gel
    • Ran 1% for 30 minutes at 100V to ensure that PCR worked
    • Everything looks good
Signal Sequence(3), IgAbc R(3), Fos(2), log-2 ladder, Fos(1), JunB(3), GCN4(3)
  • Gel
    • Ran 1.5% for 50 minutes at 100V to see if PCR product is correct length
    • Expected sizes (without VF and VR2 additions): Signal Sequence (140 bp), IgAbc (892 bp), Fos (183 bp), JunB (180 bp), GCN4 (156 bp)
    • Looks like we might have some correct inserts
    • Onwards to sequencing
  • Liquid Cultures
    • Grew up samples in 8ml of Amp/Cl media for sequencing overnight in 37C incubator (inoculated with colony PCR cell dilution):
      • Signal Sequence #2
      • Signal Sequence #3
      • IgAbc #1
      • Fos #1
      • Fos #3
      • JunB #3
      • GCN4 #2