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

• 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