Matt:LabNotes/2016-6-28

From ZhangLabWiki
Jump to navigation Jump to search

Test PA Gel Polymerization with BS(PEG)9[edit]

  • Make sure adding BS(PEG)9 doesn't inhibit polymerization
  • Check pH of gel to make sure BS(PEG)9 will work
  • Not including Acrydite/Amine linker because it is limited in quantity
    • One 100nmole order can be used 3-4 times (~$25 per use, ~$100 total)

Calculate molar concentrations in final mix[edit]

  • 5% Acrylamide = 700mM
    • 5g/100mL
    • 71.08 g/mol
  • 0.1% APS = 4.4mM
    • 100mg/100ml
    • 228.18 g/mol
  • 0.1% TEMED = 6.67mM
    • 2ul/2ml
    • 116.24 g/mol
    • 775 kg/m^3 = 775 g/L
  • Pick concentration for BS(PEG)9:
  • BS(PEG)9 = 5mM
    • 708.71 g/mol
    • After adding 465ul DMSO to stock bottle: 100mg/565ul = 177 g/L = 250mM
    • Dilute 50X in final mix just like APS and TEMED: 5mM
  • Pick concentration for Acrydite/Amine Oligo Linker
    • Ideally ~1mM in final mix but due to only having 61.1 nmol in tube have to use less
    • Resuspend 61.1 nmol in 123.2 ul = 500uM
    • Dilute 2X in final mix so final concentration: 250uM

pH of gel mix[edit]

  • BS(PEG)9 only cross-links at pH 7-9
    • APS buffers solution to pH 8-9
      • Confirmed with pH test strips

Gel Mix[edit]

  • In 1.5ml tube
  • Add APS and TEMED last
  • Let sit in 1.5ml tube for 30min
  • Positive Control
Component Volume
40% AB 199:1 Mix 12.5
10% BSA 2
H2O 81.5
5% TEMED 2
5% APS 2
Total 100
  • With BS(PEG)9
Component Volume
40% AB 199:1 Mix 12.5
10% BSA 2
H2O 79.5
5% TEMED 2
5% APS 2
BS(PEG)9 2
Total 100

Result[edit]

  • Positive control did not polymerize as much as usual/expected but there was a solid piece of gel in middle of tube
  • With BS(PEG)9 did not polymerize as well as positive control but did show signs of polymerization (more viscous and sticky)
  • pH strip indicated pH ~8
    • Forgot to include picture