Team:SPSingapore/Notebook-Week-14

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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 14 (23/8 - 29/8) ▪ Aug 27 Anaerobic Promoter    ☀ Chi Yan ☀ PCR of BL21, DH5α colonies 1 + control FP RP FPBB pNivB-R pNirB-gfp-f pNivB-R VF2 pNivB-R H20 10.25 Buffer 5 dNTP 1 FP 1 RP 1 MgCl2 2.5 Pol 0.25 Plasmid 3 1.5% gel, 130V/30min Loaded: 1kb, 100bp:    1)BL21   1)dh5α   1)control    2)BL21   2)dh5α   2)control    3)BL21   3)dh5α   3)control ▶ 1) has bands, negative has band of difference size. 2), 3), no bands ▶ rerun with new water on 28/8 Anaerobic Promoter    ☀ Yi Han ☀ PCR with hotstart kit PCR of lloendR and ivlloF1 with invP (67.9 and 60.9. used 65 deg C) H20 632ul Buffer 80ul dNTP 16ul FP 8ul RP 8ul MgCl2 32ul Pol 4ul Plasmid 20ul Total 591ul ▪ Aug 28 Anaerobic Promoter    ☀ Yi Han ☀ Ran gel for PCR on 27/8: ▶ size was correct. ▶ gel extract of PCR ▶ PCR with invlloF2 and invlloR according to previous protocol Anaerobic Promoter    ☀ Chi Yan ☀ Rerun of 27/8 with new water: Same result Anaerobic Promoter    ☀ Yi Han ☀ Ran gel for invlloF2 and invlloR on previous pcr ▶ size was correct ▶ gel extract ▶ PCR overnight with invlloF2 and invlloR. H20 280ul Buffer 40ul dNTP 16ul FP 4ul RP 4ul MgCl2 16ul Pol 4ul Plasmid 60ul Esa Quorum Sensing    ☀ Adrian ☀ Set 1 H20 110ul Buffer 40ul dNTP 16ul FP_BBP_Junk 8ul ORP_esaRBS_RBS5 8ul MgCl2 16ul GoTaq 1ul esaR_fragment 1ul Total 200ul Set 2 H20 110ul Buffer 40ul dNTP 16ul OFP_RBS5_GFP 8ul RP_BBS_Junk 8ul MgCl2 16ul GoTaq 1ul esaR_fragment 1ul Total 200ul Set 3 H20 110ul Buffer 40ul dNTP 16ul FP_BBP_Term 8ul ORP_term_esaR 8ul MgCl2 16ul GoTaq 1ul esaR_fragment 1ul Total 200ul Set 4 H20 110ul Buffer 40ul dNTP 16ul OFP_term_esaR 8ul RP_BBS_lacP 8ul MgCl2 16ul GoTaq 1ul esaR_fragment 1ul Total 200ul Buffer 4 2 BSA 2 plasmid 2.8 EcoRI 1 Spe1-HF 1 Anaerobic Promoter    ☀ Yi Han ☀ 1) PCR of inv plasmid with llostartF / LLOendBBsuffix: H20 632ul Buffer 80ul dNTP 16ul F/R primer 8ul/8ul MgCl2 32ul GoTaq 4ul template 20ul 2) Ran gel for PCR of invlloF3/invlloR ▶ no bands ▶ Ran gel to do gel extraction for invlloF1/inv110R again PCR with invlloF2/invlloR using template from gel extract. H20 260ul Buffer 40ul dNTP 16ul F/R primer 4ul/4ul MgCl2 16ul GoTaq 2ul template 60ul ▶ no bands when gel extraction 3) PCR with inv and placGFP for lloendR, invF1 H20 276ul Buffer 40ul dNTP 16ul F/R primer 4ul/4ul MgCl2 16ul Q solution 40ul template 10ul 4) PCR with above template for lloendR/invF2. H20 80ul Buffer 20ul dNTP 16ul F/R primer 2ul/2ul MgCl2 8ul template 80ul(800ng) ▶ Ran gel ▶ Size correct ▶ purity, yield, 56.6ng/ul in 50ul