Team:SPSingapore/Notebook-Week-4

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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 4 (14/6 - 20/6) ▪ June 14 Anaerobic Promoter    ☀ Yi Han ☀ PCR ▶ RP_XhoI_GFP and FP_KpnI_GFP with GFP-3 as template ▶ for 7 reactions with control Mastermix * 8 PCR buffer 80ul Primers 0.8ul/0.8ul DNA polymerase 4ul dH2O 494ul Templates 38.75 Digest more plasmid (Esa plasmid) 4 rxns ( KpnI/XhoI digest) 50ul each Buffer 20ul KpnI 2ul XhoI 2ul DNA 34.8ul H20 141.2ul ▪ June 15 Anaerobic Promoter    ☀ Yun Ting ☀ PCR: RP_XhoI_GFP and FP_KpnI_GFP with GFP midiprep for 15 reactions with control: PCR buffer 170ul Primers 1.7ul, 1.7ul dNTP 34 ul DNA polymerase 8.5 ul dH2O 1127.1ul Templates 17ul Total 1360ul PCR protocol “GFP 1” (32 cycles) Anaerobic Promoter    ☀ Yi Han ☀ GFP PCR product -> 448ng/ul Gel extraction Qiagen gel extraction kit at MBI Esa gel band from 14/6 Nanodrop: 16.4 ng/ul, 260/280 = 2.60 Plasmid construction RE digest (KpnI, XhoI) 3 Replicates of the following: DNA 2.6ul Buffer 5ul H2O 41.5ul KpnI 0.5ul XhoI 0.5ul Ligation (2x reaction) 40ul 10X T4 DNA ligase buffer 4ul Vector DNA (16ng/ul) 100ng -> 6.25ul insert DNA (75ng) 12 ul T4 DNA Ligase 2ul H2O 16ul ▶ Note: ALL digested DNA in tube labeled lacGFP.ligation was used ▶ Nanodrop of ligation: 1114.0ng/ul. 260/280 = 3.7 ▪ June 16 Anaerobic Promoter    ☀ Yi Han ☀ Transformation of ligated esa-GFP plasmid into DH5\alpha cells DH5alpha, unlabelled brown vial inside DH5\alpha box at -80 ▶ Plates spread at 1140h ▶ LB + chloroamphenicol      4x (100ul)      10x (40ul)      20x (20ul)      LB - 20x (20ul) Remaining transformed cells (~220ul) are kept at 4C ▶ labeled as placGFP in brown tube ▪ June 17 Anaerobic Promoter    ☀ Yi Han ☀ only 4x placGFP plate has colonies (7) ▶ spread one new LB + chlor plate with 200ul of transformed bacteria ▶ picked 6 colonies to grow for miniprep in LB + chl liq media ▶ Conclusion: Protocol works but optimization for increase vol needed Cloning of synparts in amp vector ▶ comes as 4mg dry DNA ▶ +40ul of DI/RNAse free H20 ▶ for transformation, 150ml wed Miniprep of placGFP followed by RE digest (Kpn, XhoI): 50 ul total Buffer 2.5 KpnI 1 XhoI 1 DNA 10 H20 120.25 Colony PCR for synparts ▶ Failed ▶ No specific bands produced ▶ Might need optimization Invasin + Listerolysin    ☀ Yan Ting ☀ Subculture of HEK 293 ▶ P4 -> p5 ▶ grow/split into 150mm dish for freezing on sat (20/6)      30ml DMEM + 1ml cells ▶ 90mm dish for maintenance (buffer)      10ml DMEM + 30ul cells ▶ Cells combined from 3 90mm dishes ▪ June 19 esa Quorum Sensing    ☀ Yi Han ☀ RE digest (XhoI, KpnI) of esa and GFP esa Quorum Sensing    ☀ Yun Ting ☀ Gel electrophoresis (100V, 40min). ▶ Lane 2: gpf: no bands ▶ Lane 3: 100bp ladder ▶ Lane 4: blank ▶ Lane 5: esa: 2 bands ▶ Lane 6: 1kb ladder ▶ Lane 7:blank ▪ June 20 Invasin + Listerolysin    ☀ Yan Ting ☀ Freezing of HEK293 ▶ P6 ▶ cells from a 150 mm dish and 90mm dish