Team:SPSingapore/Notebook-Week-16

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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 16 (6/9 - 12/9) ▪ Sep 6 Anaerobic Promoter    ☀ Yi Han ☀ Ligation of placgfp insert into pSB1C3 Cut vector 20 cut insert 20 T4 ligase buffer 5 Enzyme 2 PCR F2 from F1 dH2O 465 Buffer 60 MgCl2 24 dNTP 24 Primers 12/12 GoTaq 3 Template 65 Total 600 ▶ extracted above product ▪ Sep 7 Anaerobic Promoter    ☀ Chi Yan ☀ No colonies for placgfp pSB1C3   Plated remaining bacteria onto chl plates   Redo RE (Yi Han 5/9) 2h15min   Ligate at 16 degrees overnight PCR of F2 using:   1. invlloF3new / lloendR to make F3   2. invlloF4/ lloendR to make F4 dH2O 200 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 GoTaq 1 Template 16.7 Total 200 ESA Quorum Sensing    ☀ Kenneth ☀ Colony PCR dH2O 13.5 Buffer 5 MgCl2 2 dNTP 2 Primers 1/1 GoTaq 0.5 Total 25 Anaerobic Promoter    ☀ Yi Han ☀ Transformation of bacteria using remaining ligated placgfp Anaerobic Promoter    ☀ Chi Yan ☀ PCR to get invF1, Ta 58degrees, 3 min extension time   Primers invlloF1/lloendR dH2O 268 Buffer 40 MgCl2 16 dNTP 16 Primers 4/4 Taq 2 Template 10 Total 400 Test invllloF4/lloendR with 10 cycles 45degrees Ta dH2O 35.7 Buffer 5 MgCl2 2 dNTP 2 Primers 0.5/0.5 Taq 0.125 Template 4.2 Total 50 Result when gel was run - smear ▪ Sep 8 Anaerobic Promoter    ☀ Yi Han ☀ Gel extract of F1 Making more F2 dH2O 64 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 80 Total 200 Ran gel for F2, gel extracted PCR to get F4 (invlloendR/ invlloF4)   10 cycles of Ta 35, 45, 50 degrees   30 cycles of Ta at 60 degrees dH2O 39.5 Buffer 20 MgCl2 4 dNTP 4 Primers 2/2 Taq 1 Template 40 Total 100 ▶ Results: smear in gel ESA Quorum Sensing    ☀ Kenneth ☀ Ran gel for La, C T and L Anaerobic Promoter    ☀ Yi Han ☀ PCR with F3new/ lloendR on F2 dH2O 142 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 17 Total 200 PCR with F4/lloendR using F2 dH2O 70.5 Buffer 10 MgCl2 4 dNTP 4 Primers 2/2 Taq 0.5 Template 10 Total 100 Cycle conditions for F3new fragments:   5 cycles for gradient Ta 50-60degrees   30 cycles for gradient Ta from 55-65 degrees ▶ Result, smear for all Invasin + Listerolysin    ☀ Yun Ting ☀ Prefixplacinvpcr 2.3, with new F2’ and RP2 Cycle conditions:   5 cycles of Ta 48 degrees   30 cycles of Ta 62 degrees (59, 61, 62, 64) L4 template: PCR product 1.4.10 5-8 template: gel elute 1.1 No bands but see ‘9.8 pcr 2.3 + 1.4’ Lane 2-5 are tube 1, 4, 5, 8 seems to have 2.8kb band Could be template Run negative control with no template next time placinvPCR 1.5 dH2O 157 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 1 Total 200 ▶ Thermocycler conditions for above:    10 cycles of Ta of 48degrees    30 cycles of Ta of 8.3, 59.3 (much brighter) ▶ Tube 3 was much brighter than rest, 3-4 had no product ▪ Sep 9 Anaerobic Promoter    ☀ Yi Han ☀ PCR for F3new/lloendR and F4/lloendR from F2 fragment ▶ 1st step, 10 cycles gradient from 45-60 degrees Ta. ▶ 2nd Step, 30 cycles of PCR wt Ta 60 degrees Ran gel, smear for all Repeated F3new/lloenR using same reagent ratio 5 cycles 40-46 gradient 5 cycles 40-46 gradient 30 cycles Ta 60 degrees Invasin + Listerolysin    ☀ Yun Ting ☀ Prefix placinv PCR 2.4 Thermocycler ▶ 8 cycles at Ta = 48 ▶ 30 cycles of Ta 55, 56,2, 57, 58.7, 59.6, 61.8 and negative control dH2O 30 Buffer 40 MgCl2 16 dNTP 16 Primers 4/4 Taq 2 Template 8 Total 400 Anaerobic Promoter    ☀ Yi Han ☀ RE of psB1C3-BBa_J04450 with EcoRI/PstI (1000ng) RE of placgfp with EcoRI/PstI (1000ng) for 3 hours placgfp Template 16 Buffer 5 EcoRI 0.5 PstI 0.5 H2O 28 Total 50 psB1C3-BBa_J04450 Template 23 Buffer 5 EcoRI 0.5 PstI 0.5 H2O 21 Total 50 Anaerobic Promoter    ☀ Chi Yan ☀ Inoculated 3mL x8 tubes pSB1C3 in LB+chl Ran gel for YH 9/9 RE digest in 1% gel 1kb 100bp 2 lanes vector space, 2 lanes insert placgfp was not amplified -> gel extract pSB1C3 PCR to make    1. prefix-pNirB-inv-suffix invlloF3/invendBBSuffix    2. F4 invlloF4/invendBBsuffix    3. Prefix placgfp-suffix: BBPrefix-placgfp-F/GFPBBSuffix-Rnew 1: F: overlap Tm 56.4    whole 66.7 R:overlap 48    Whole 66.5 2: F: overlap Tm 50    whole 63 R:overlap 48    Whole 66.5 3: F: overlap Tm 55    whole 68 R:overlap 51.7    Whole 65.6 dH2O 141 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 18 Total 200 ▶ Gradient 42-55 degrees 7 cycles, 60 degrees 30 cycles, ramp rate 20% ▶ 3. Correct RE pSB1C3-red ExoRI/PStI same as Yi Han 9/9 2.30pm Invasin+Listerolysin    ☀ Yan Ting ☀ Ran gel for Yun Ting 9/9 PCR ▪ Sep 10 Anaerobic Promoter    ☀ Chi Yan ☀ PCR of invD with invlloF1 and invend BB suffix   F: Overlap Tm 57.5     whole 65.6   R: Overlap 48.4     whole 66.5 dH2O 148 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 4.6 Total 200 ▶ Gradient 42-55 degree 7 cycles 60 degrees 30 cycles Ran gel for Chi Yan 9/9 1,2 smear, 3 correct size Inoculate placgfp in 3mL x6 tubes in LB+chl Gel extract prefplacgfp-suff from Chi Yan 10/9 PCR to extend prefix and suffix with Juunk sequences using FP_BBP_Junk, RP_BBS_Junk dH2O 148 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 11 Total 200 ▶ Ran gel to check size correct. PCR direct purification of 180ul reation: 160ng/ul in 50ul pSB1C3 was incompletely digested miniprep SB1C3 RE pSB1C3 Junk=pref=placgfp-suf-JUNK with EcoRI/PstI PsB1C3 DNA 1000ng 6.6 Buffer 10 EcoRI 1 PstI 1 water 81.4 Total 100 placgfp DNA 300ng 2 buffer 3 EcoRI 0.3 PstI 0.3 Water 24.4 Total 30 1. PCR of invlloF1invendsuffix with invlloF2 and invendsuffix to make invlloF2 end suffix 2. PCR of F2 6/9 with invlloF6, invend BBsuffix 3. PCR ofo F2 6/9 with invlloF3new, invendBBsuffix Part 1 4. PCR of F3 6/9 with invlloF2, invendBBsuffix dH2O 145 Buffer 20 MgCl2 8 dNTP 8 Primers 2/2 Taq 1 Template 14 Total 200 ▶ 7 cycles 44-5 degrees 30 cycles 60 degrees ramp rate 20% ▶ Cycle 95degrees 15min-> 94deg 30s X deg 40s 72deg 3min 50s 72 deg 10min Invasin + Listerolysin    ☀ Yun Ting ☀ Prefix placonvPCR 2.5 8 reactions template as 1.4.10 Thermocycler 8 cycles Tm 30-40 degrees 8 cycles 40-50 degrees 30 cycles Tm = 50-60 degrees No bands prefix-placinvPCR 2.6 and 1.6 from each and negative control template for 2.6 is 2.4 (indistinct 2.8k band) For 1,63 plasmid was 300ng/100ul Themocycler for 25 cycles at Ta 40-55 degrees (40, 44, 40, 553) Ramp speed 20% Gel elution of PCR 1.5 ½ Gel run with bigwells comb Anaerobic Promoter    ☀ Yi Han ☀ 1. Ran gel for YT, band observed for F1 fragment Gel extracted 2. miniprepped plac plasmid in esa backbone 3. gel extract pSB1C3 fragment (E/P) and (E/P) plagfp fragment Ligation of pSB1C3 to placgfp 5:1 Vector (70ng) 2.2 insert (140ng) 5 Buffer 2 Enz 0.2 H2O 10.6 Total 20 7:1 vector (70ng) 2.2 Insert (196ng) 7 Buffer 2 Taq 0.2 H2O 8.6 Total 20 Invasin+Listerolysin    ☀ Yun Ting ☀ PCR 2.7 Template is 2.4.4 Thermocycler for 17 cycles at Ta 49, 55, 62, 65 13 cycles at Ta 42, 48, 56, 60, extension time 3min dH2O 240 Buffer 30 MgCl2 12 dNTP 12 Primers 3/3 Taq 1.5 Template 1 Total 300 ▪ Sep 11 Anaerobic Promoter    ☀ Chi Yan ☀ 1. gel extract PCR product from 10/9 PCRs to make    A F1invsuffix: inv D plasmid on invlloF1/invendBB suffix 800ul    B F2invsuffix: F1invsuffix on invlloF2/invendBB suffix 400ul    C F3invsuffix: F2invsuffix on invlloF3new/invendBB suffix 100ul    D F6invsuffix: inv D plasmid on invlloF6/invendBB suffix 100ul A B C, D dH2O 617.7 293 66.5 Buffer 80 40 10 MgCl2 32 32 4 dNTP 32 32 4 Primers 8/8 8/8 1/1 Taq 4 4 0.5 Template 18.3 18.3 12.5 Total 800 800 100 ▶ 7 cycles gradient 49-58 degrees ▶ 30 cycles 60 degrees ▶ A : 6 at 53.8, 6 at 55, 4 at 56degrees    B : 4 at 49.1, 4 at 49.3    C : 1 each at 49.1 49.8 50.6 51.6 52.7 53.8    D : 1 each at 57.5, 57.8 ▶ Transformed 5ul of each ligation reaction for placgfp 5:1 and 7:1 into BL21 RE of junk-pref-placgfp-suff-junk with EcoRI/PstI 500ng of DNA 3.1 Buffer 5 EcoRI 0.5 PstI 0.5 H2O 40.9 Total 50 Ran gel for 2. Invasin + Listerolysin    ☀ Yun Ting ☀ PCR 2.8 300ul reaction mix template 1.3 1.4 Thermocycler: ▶ 10 cycles 51, 56, 60 3min annealing ▶ 20 cycles +4, 40s annealing Anaerobic Promoter    ☀ Yi Han ☀ PCR with GO Taq kit Control reaction with no primers dH2O 14.25 Buffer 5 MgCl2 2.5 dNTP 1 Template 2 Taq 0.25 Total 25 F2 from F1 invend suffix dH2O 204.2 Buffer 80 MgCl2 40 dNTP 16 Primers 16/16 Taq 4 Template 23.8 Total 400 Anaerobic Promoter    ☀ Chi Yan ☀ Purifying RE of Junk-prefix-placgfp-suffix-junk PCR with Go Taq kit A,B dH2O 5.25 Buffer 15 MgCl2 7.5 dNTP 3 Primers 3/3 Taq 0.75 Template 37.5 Total 75 ▶ Annealing temp of 59 degrees Anaerobic Promoter    ☀ Yi Han ☀ Ran PCR for F2 ▶ 5 cycles anneal temp 50 ▶ 30 cycles anneal temp 58 Ran gel, gel extract F2 ▪ Sep 12 Anaerobic Promoter    ☀ Chi Yan ☀ PCRs to make    A F2invsuffixP1: F1invsuff on invlloF2/invendBB suffixP1 200ul    B F6invsuffix: F2invsuffix on invlloF6/invendBB suffix 75ul    C F6invsuffixP1: F2invsuffix on invlloF6/invendBB suffixP1 100ul    D F1endsuffix: F2invsuffix on invlloF1/invendBB suffix 100ul A B C D dH2O 155 25.2 47.5 8.32 Buffer 20 7.5 10 2.5 dNTP 8 3 4 1 Primers 2/2 0.75/0.75 1-Jan 0.25/0.25 Taq 1 0.375 0.5 0.125 Template 12 37.5 31 12.5 Total 200 75 100 25 Anaerobic Promoter    ☀ Yi Han ☀ PCR F2 from F1 dH2O 155.1 Buffer 20 dNTP 8 Primers 2/2 Taq 1 Template 11.9 Total 200 Ligation of new placgfp ratio 5:1 as of 10/9 Anaerobic Promoter    ☀ Chi Yan ☀ Colony PCR of placgfpSB1C3 with FP_KpnI-GFP,GFP-Suffix R 25 cycles, 60degrees Ta ESA Quorum Sensing    ☀ Adrian ☀ GFPThing EcoRI/PstI Buffer 2 GFP thing 13.3 EcoRI 1 PstI 1 dH2O 2.7 Total 20 ▶ OFP_term_esaR & ORP_esaRBS_RBSS -> 1kb ▶ OFP_RBBS_GFP & ORP_Term_esaR -> 3kb Anaerobic Promoter    ☀ Chi Yan ☀ PCRs to make Prefix-pNirB-inv-suffix   E: F1endsuffix as template->F2invendsuffix with invlloF2/invendsuffix (400ul)   F: F2endsuffix 12/9 as template-> F2invendsuffix with invllloF2/invendsuffix (100ul)   G: F2endsuffix as template-> F6invendsuffix with invlloF6/invendsuffix (100ul)   H: F2invendsuffixP1 12/9 as template-> F2invendsuffix with invlloF2/invendsuffix (75ul)   I: F2invendendsuffixP1 12/9 as template -> F6invendsuffix with invlloF6/invendsuffix (75ul) Cycle conditions    95degrees 15min    5cycles    94degrees 30 seconds    gradient 49-58 30 seconds    72degrees 3min    20cycles    94degrees 30 seconds    60 30 seconds    72degrees 3min E F,G H,I Buffer 40 10 7.5 dNTPs 16 4 3 Primers 4/4 1/1 0.75/0.75 Template 23.8 30.7 24.5 Taq 2 0.5 375 H2O 310.2 52.8 38.2 Total 400 100 75 ▶ E1-8 49.1d egrees ▶ E9-16 49.3 degrees ▶ F1-4 57.8 degrees ▶ G1-2 57.5 degrees ▶ G3-4 57.8 degrees ▶ H1-3 55 degrees ▶ I1-3 55 degrees Invasin + Listerolysin    ☀ Yi Han & Chi Yan ☀ PCR 2.9 for prefix-plac-Inv-suffix, 6 rxn. Primers used are FP2’_prefixplac and RP_Invendsuffix. . Template is PCR product 2.8.1. Thermocycler 10 cycles at Ta=,56,58,60deg, annealing time=3min; then 20 cycles at Ta= 60,62,64deg, annealing time=40s. Ramp speed =20%. Smear. PCR2.10 using PCR2.8 protocol. Faint 2.8kb band visible amongst smear. Ran gel with gel extracted product from 2.10 (supposed 66ng/ul with poor 260/280 ratio). No visible band.