Team:SPSingapore/Notebook-Week-15

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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 15 (30/8 - 5/9) ▪ Aug 30 Anaerobic Promoter    ☀ Yi Han & Chi Yan ☀ PCR with above template using lloendR/invF3 H20 140ul Buffer 20ul dNTP 8ul F/R Primer 2ul/2ul MgCl2 8ul Template 80ul(800ng)(250ng/tube) Taq 1ul ▶ 4 tubes. Nanodrop (1224ng/ul). 1 tube kept for running gel 3 tubes purify from suspension using Thermo(118.7 ng/ul overall). use at least 20ul elution buffer ▶ Fusion PCR with hotstartaq DNA pol Primers invlloF3 + lloEndBBSuffix 1. 200ng/tube for each template 2. 100ng/tube 3. 200ng/tube for each template + Q solution ▶ First run 10 cycles without primers. Extension for 30s Then add primers, change settings to 95 deg C 10sec start, run 30 cycles. Extension for 4.46. Ta of 65 deg C Miniprep 3 tubes of invD plasmid. 218ng/ul of 150ml 1 2 3 PCR mix 200ng(1) 100ng(2) 200ng + Q solution(1) Buffer 20ul 20ul 20ul dNTP 8ul 8ul 8ul MgCl2 8ul 8ul 8ul F/R Primer 2ul/2ul 2ul/2ul 2ul/2ul Taq 1ul 1ul 1ul Q solution - - 40ul lloendF1 / lloBBsuffix 3.5ul 1.75ul 3.5ul F3 4ul 2ul 4ul H20 151ul 155ul 151ul Total = 200 ul/each ▶ Ran gel: smear for all ▶ After consulting Elvin (clear band of correct size should be produced) Reran above Fusion PCR for condition 2, with Ta at 58 deg C, extension time 5min ▶ Remade more Fragment 3 as with earlier reaction ▪ Aug 31 Anaerobic Promoter    ☀ Chi Yan ☀ Ran gel for fusion PCR and Fragment 3 2 large 2% gels, 120V 60min:   (1) Fragment 3.     100bp, 1kb with F3 contamination, F3 1kb, 100bp,   (2) 4.4kb     1kb, 100bp, 8 lanes & 4 kb, 1kb, 100bp Yi Han -> Smears for F3 and 4.4kb fragment. Oh no!   Redo PCR for F3 with less template, less primer   previously 250ng/tube 50, 100, 150ng   primer 0.1uM concentration, 0.1, 0.05 uM Permutations: 0.1uM primer 50ng 0.1uM primer 100ng 0.1uM primer 150ng 0.05uM primer 50ng 0.05uM primer 100ng 0.05uM primer 150ng Buffer 20ul dNTP 8ul MgCl2 8ul Primers (0.1uM)->2/2||(0.05uM)->1/1 Template (50ng/tube|3.57ul)|| (100ng/tube|7.14ul)|| (150ng/tube|10.7ul) Taq 1ul H20 158/155/151 ul ▶ 28 cycle (Ta is 58, extension of 3min) did (1), (2), (4) ▶ 2 1% gels double row Invasin + Listerolysin    ☀ Yun Ting ☀ PCR H20 326 ul Buffer 40ul MgCl2 16ul Primers 4ul/4ul dNTP 16ul Template 8ul Total 400ul PCR for placInv, 8 rxn. Primers used are FP1 placInv and lloend-R. Template is InvLLO plasmid miniprep (115ng/ul). No bands. H2O 310 Buffer 40 dNTPs 16 MgCl2 16 Primers 4+4 Template 8 Taq 2 Anaerobic Promoter    ☀ Yi Han ☀ Ran gel, only primer dimers. Lowering concentration of template or primer did not help -> no primer Purified F1 (32.6ng/ul) and F2 (63.1ng/ul) from suspension ▶ F2 and F3 were PCRed out using same reaction conditions as 30/8 ▶ F1/F2 template total 1000ng ESA Quorum Sensing    ☀ Kenneth ☀ Gel extraction of the sequences: ▶ Term Tube 1: 0.237g -> 25.2ng/ul ▶ EsaR Tube 2: 0.182g -> 164.2ng/ul ▶ EsaRBS Tube 3: 0.202g -> 10ng/ul ▶ GFP Tube 4: 0.224g -> 118ng/ul dH2O 92.56ul Buffer 40ul MgCL2 16ul dNTP 16ul FP_BBP_Term 8ul RP_BBS_lacP 8ul GoTaq 1ul term (400ng) 16ul EsaR (400ng) 2.4ul Total 200ul dH2O 72.82ul Buffer 40ul MgCL2 16ul dNTP 16ul FP_BBP_Term 8ul RP_BBS_lacP 8ul GoTaq 1ul term (400ng) 40ul EsaR (400ng) 3.38ul Total 200ul Anaerobic promoter Brown    ☀ Chi Yan ☀ Redo 29/8 to get inv F1 Tm 58degrees, 3 min extension, 35 cycles, 2ulTaw labelled CY ▪ Sep 1 Anaerobic Promoter    ☀ Yi Han ☀ Ran gel from CY 31/8 -> gel extract, PCR to get F2 with invlloF2/lloendR dH2O 115 Buffer 20 MgCL2 8 dNTP 8 Primers 2/2 GoTaq 0.5 Template 45 Total 200 ▶ CY ran the gel from above to get F2 ▶ YH verified size was correct despite smear being present and gel extracted band of appropriate size ▶ PCR of F3 from F2 using invlloF3 and BBprefix-invasin Suffix dH2O 125 Buffer 20 MgCL2 8 dNTP 8 Primers 2/2 GoTaq 0.5 Template 35 Total 200 Invasin + Listerolysin    ☀ Yun Ting ☀ Reran PCR from 31/8 for placInv, 8 rxn. Used thermogradient for annealing temperature – higher Ta near 65deg seems to work better. ▶ Ran gel with 20ul per well – bands just below 3kb. ▶ Cut out 7 bands, placed in 2 gel extraction columns. Eluted with H2O that has been warmed to 55deg. Poor yield– 15ng/ul. Reran PCR from 31/8 for placInv, 4 rxn. Adjusted thermogradient for Ta= 60, 62, 65, 67deg for 40cycles O/N. Generally faint bands, especially at 60deg. dH2O 169 Buffer 20 MgCL2 8 dNTP 8 Primers 2/2 GoTaq 1 Template 2 Total 200 ▪ Sep 2 Anaerobic Promoter    ☀ Chi Yan ☀ Ran gel from YH 1/9 F3 and BBPrefix-inv-BBsuffix     1% 100v 45min     1kb 100bp F3 BBPre-inv-BBsuffix 100bp 1kb Result: Smear Ran gel for Cy 31/8 1% 100v 45min Gel extract Anaerobic Promoter    ☀ Yi Han ☀ PCR to get F1 as of 29/8 Pcr to get F2 as with 1/9 using gradient PCR Ran gel @ 4pm, Ran PCR to get F3, inv fragment Invasin + Listerolysin    ☀ Yan Ting ☀ Gel extraction nanodrop results 1: 9.8ng/ul 2: 3.0ng/ul Anaerobic Promoter    ☀ Yi Han ☀ PCR of placgfp with BBPrefixplacGFP-F/BBSuffixGFP-R dH2O 100 Buffer 20 MgCL2 8 dNTP 8 Primers 2/2 GoTaq 1 Template 60 Total 200 Repeat PCR for inv fragment as of 1/9 Ran gel for above samples ▶ No PCR product for placgfp ▶ Smear for invasin fragment ▶ Band of correct size for F3 - gel extract ▶ Amplify more F3 from F3 PCR Reaction ▶ Template-> gel extracted F3 ▶ Primers invlloF3/lloendR dH2O 298 Buffer 40 MgCL2 16 dNTP 16 Primers 4/4 GoTaq 2 Template 20 Total 400 Invasin + Listerolysin    ☀ Yun Ting ☀ PCR 1.4 for placInv, 10 rxn. Primers used are FP1 placInv and lloend-R. Template is InvLLO plasmid miniprep (115ng/ul). Ta= 58-64deg, Extension time=3.5min. ▪ Sep 3 Anaerobic Promoter    ☀ Chi Yan ☀ Miniprep of placgfp then gradient PCR for placgfp following YH 2/9 with BBPRefixplacgfp-F and BBSuffixGFP-R dH2O 288 Buffer 40 MgCL2 16 dNTP 16 Primers 4/4 GoTaq 2 Template 35 Total 400 Gradient 58-63 degrees Chose: 58, 58.4, 58.8, 60, 60.7, 61.3, 62.4, 62.9 Anaerobic Promoter    ☀ Yi Han ☀ Ran gel for F3 from F3 => Smear PCR for fusion fragment   1:1 ratio of template   500ng Front fragment (5ul)   500ng back fragment (5ul) Buffer 20 MgCL2 8 dNTP 8 Primers 2/2 Taq 1 H2O 44 Total 100 Anaerobic Promoter    ☀ Chi Yan ☀ Yun Ting's PCR Nanodrop     1. 811     2. 722     3. 730     4. 710     5. 727 Ran gel for Yun Ting’s PCR (5/7 and cut bands), Yi Han's 3/9 fusion pcr (no bands) 100v 40 min ESA Quorum Sensing    ☀ Kenneth ☀ PCR purification of 31/8 Anaerobic Promoter    ☀ Chi Yan ☀ PCR to get F2 and F3 invlloF2/lloendR dH2O 123 Buffer 20 MgCL2 8 dNTP 8 Primers 2/2 GoTaq 1 Template 36 Total 200 invlloF3/lloendR dH2O 306 Buffer 40 MgCL2 16 dNTP 16 Primers 4/4 GoTaq 2 Template 12 Total 400 58 degrees annealing, 42 cycles, 3min extension PCR of placgfp with BBPrefixplacGFP-F/BBsuffix GFP-R and GFP-R/BBPrefixplacgfp-F as control (25ul) Half of normal MgCl2 concentration used, rediluted all 3 primers Gradient PCR 55 to 65 degrees BBPrefixplacGFP-F/BBsuffix GFP-R dH2O 218.2 Buffer 30 MgCL2 6 dNTP 12 Primers 3/3 GoTaq 1.5 Template 26.3 Total 200 GFP-R/BBPrefixplacgfp-F dH2O 18.2 Buffer 2.5 MgCL2 2.5 dNTP 1 Primers 0.25/0.25 GoTaq 0.13 Template 2.2 Total 25 Inoculated 4 tubes 3mL LB + ch1 placgfp Anaerobic Promoter    ☀ Yi Han ☀ Ran gel for F2 (product), F3 (smear) and placgfp (no product)   Smear for F3 faint band for F2   gel extract F2 PCR using GoTaq kit for F3 and inv fragment dH2O 53.5 Buffer 10 MgCL2 4 dNTP 4 Primers 4/4 GoTaq 0.5 Template 10 Total 100 PCR with hotstart and inv 5 cycles with Ta 46 first 30 cycles overnight at Ta 60 dH2O 27.5 Buffer 4 MgCL2 4 dNTP 1 Primers 1/1 GoTaq 0.5 Template 2 Total 50 Remake F2 dH2O 144 Buffer 20 MgCL2 4 dNTP 4 Primers 2/2 GoTaq 1 Template 15 Total 200 Remake F1 dH2O 333 Buffer 40 MgCL2 8 dNTP 8 Primers 4/4 GoTaq 2 Template 9 Total 400 ▪ Sep 4 Invasin + Listerolysin    ☀ Yun Ting ☀ PCR prefix-placinv for 10 reactions dH2O 348 Buffer 40 MgCL2 16 dNTP 16 Primers 4/4 GoTaq 2 Template 10 Total 400 Ran gel at 110V, 1h. Marker was not distinct. No bands. PCR 2.1 for prefix-plac-Inv-suffix, 8 rxn. Primers used are FP2_prefixplac and RP_Invendsuffix. For tubes 1-4, template is PCR product 1.4.10 (i.e. tube 10 from PCR reaction 1.4). For tubes 5-8, template is gel extracted PCR product 1.2 (i.e. gel extract from PCR reaction 1.2). Ta= 59, 61, 62, 64deg. No bands. H2O 310 Buffer 40 dNTPs 16 MgCl2 16 Primers 4+4 Template 8 Taq 2 PCR 2.2 for prefix-plac-Inv-suffix, 4 rxn. Primers used are FP2_prefixplac and RP_Invendsuffix. Template is PCR product 1.4.10 (i.e. tube 10 from PCR reaction 1.4). Thermocycler 5 cycles at Ta=27deg (Ta of FP2), then 30 cycles at Ta= 58 & 60deg. No bands. ▪ Sep 5 Anaerobic Promoter    ☀ Yi Han ☀ Ran gel for inv fragment, F1, F2   Smear for inv fragment, product for Fa and F2 gel extract   Trying again to make inv fragment   5 cycles at 46 degrees, 30 cycles at 59 degrees Inv fragment dH2O 3.75 Buffer 5 MgCL2 2 dNTP 2 Primers 1/1 GoTaq 0.25 Template 15 Total 50 PCR for placgfp   5 cycles at Ta 40 degrees   30 cycles at Ta 30 defrees and 60 degrees (12 tubes from 200ul of PCR reaction mix) dH2O 124 Buffer 20 MgCL2 8 dNTP 8 Primers 4/4 GoTaq 1 Template 31 Total 200 PCR for F2 dH2O 125 Buffer 20 MgCL2 8 dNTP 8 Primers 4/4 GoTaq 1 Template 20 Total 200 Invasin + Listerolysin    ☀ Yun Ting ☀ PCR 2.2 PrefiplacInv for 2 reactions Addition of 5 cycles at Ta=27 degrees before 30cycles with (Ta 58 & 60 degrees) Anaerobic Promoter    ☀ Yi Han ☀ Results of PCR -> Faint band of F2-> gel extract   no product still smear for inv fragment   Clear band for placgfp-> gel extract (no amplification) PCR to make inv fragment from F3 and PCR for more F2   5 cycles at 30, 5 cycles at 40, 30cycles at 55 degrees Ta Invasin fragment dH2O 105 Buffer 20 MgCL2 8 dNTP 8 Primers 4/4 GoTaq 1 Template 50 Total 200 ▶ Ran gel, result is no amplification RE of linearised backbone of pSB1C3 and placgfp fragment with BB prefix and Suffix 125 ng linearised backbone (2kb) 5 10X buffer 2 EcoRI 0.2 PstI 0.2 H2O 12.6 Total 20 250 ng pcr product 7.6 10X buffer 2 EcoRI 0.2 PstI 0.2 H2O 10 Total 20 Ran RE for 3 hours PCR to make more F1 dH2O 293 Buffer 40 MgCl2 8 dNTP 8 Primers 4/4 GoTaq 1 Template 17 Total 400 ESA Quorum Sensing    ☀ Adrian ☀ RE digest: EsaR thing 29.1 10X buffer 4.5 EcoRI 1 SpeI 1 BSA 4.5 H2O 4.9 Total 45 GFP Thing 13.3 10X buffer 2 EcoRI 1 PstI 1 H2O 2.7 Total 20 Ligation Vector 2 Insert 1 4 Insert 2 5.58 10X buffer 2 Ligase 1 H2O 5.42 Total 20 ▶ 5ul of ligated product was transformed into dH5 alpha