Team:SPSingapore/Notebook-Week-4

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  • Week 1 (24/5 - 30/5)
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  • Week 4 (14/6 - 20/6)
  • Week 5 (21/6 - 27/6)
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  • Week 14 (23/8 - 29/8)
  • Week 15 (30/8 - 5/9)
  • Week 16 (6/9 - 12/9)
  • Week 17 (13/8 - 17/9)

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