Team:SPSingapore/Notebook-Week-3

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• NOTEBOOK
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• PROTOCOL
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• ENTRIES
  • Week 1 (24/5 - 30/5)
  • Week 2 (31/5 - 6/6)
  • Week 3 (7/6 - 13/6)
  • Week 4 (14/6 - 20/6)
  • Week 5 (21/6 - 27/6)
  • Week 6 (28/6 - 4/7)
  • Week 7 (5/7 - 11/7)
  • Week 8 (12/7 - 18/7)
  • Week 9 (19/7 - 25/7)
  • Week 10 (26/7 - 1/8)
  • Week 11 (2/8 - 8/8)
  • Week 12 (9/8 - 15/8)
  • Week 13 (16/8 - 22/8)
  • Week 14 (23/8 - 29/8)
  • Week 15 (30/8 - 5/9)
  • Week 16 (6/9 - 12/9)
  • Week 17 (13/9 - 18/9)

Research Notebook

Week 3 (7/6 - 13/6) ▪ June 8 Anaerobic Promoter    ☀ Yi Han ☀ Miniprep of gfp plasmids, preparation of samples for sequencing ▪ June 9 Anaerobic Promoter    ☀ Yi Han ☀ All gfp plasmids had a correct sequence Yanting kept glycerol stock for all, and inoculation of 3mL of gfp3 into 100mL LB+amp for midiprep ▪ June 10 Anaerobic Promoter    ☀ Yi Han & Yun Ting ☀ Midiprep of gfp plasmid PCR of gfp with KpnI-gfp and XhoI-gfp primers ▪ June 11 esa Quorum Sensing    ☀ Yun Ting & Duy ☀ Nanodrop of gfp product-> 595.5ng/ul RE digest of EsaR vector with KpnI/XHoI Gel electrophoresis at 1000V for 30min ▪ June 12 esa Quorum Sensing    ☀ Yun Ting ☀ Gel extraction using Promega binding solution to melt gel, followed by thermo scientific kit Poor results: EsaR 1.9ng/uL, gfp 3.6ng/ul Heated EB to 70degC and incubated on column for 30min Poor results: EsaR: 4.3ng/ul, Gfp: 2.8bg/ul esa Quorum Sensing    ☀ Adrian ☀ Gel extraction optimisation Hypothesised that the Binding buffer has a problem/DNA does not bind to column    1: 2X promega binding buffer volume    2: Increase incubation time for binding to 5min Switch binding buffer to that of thermo scientific PCR purification kit Use sodium acetate if available? TO facilitate stronger binding to column Results    1: ~10ng/ul    2: ~9ng/ul    not succesful    further optimisation-> warm buffer, incubate for 5min    elute in 30/20ul smaller volumes ▪ June 13 esa Quorum Sensing    ☀ Yi Han ☀ 1: Thermoscientific miniprep columns with 2XThermoscientific binding buffer 2: Thermoscientific PCR purification kit 2X buffer 3: Promega kit 2X buffer Protocol failed despite heating EB, increasing incubation time and eluting 2X yields < 10ng/ul