Team:SPSingapore/Notebook-Week-9

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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 9 (19/7 - 25/7) ▪ July 19 Invasin + Listerolysin    ☀ Yi Han ☀ Inoculation of positive colonies in liquid culture. 3mL and shaking incubation. inoculated colonies C,D for invasin plasmid in 3mL LB+amp at 1.30pm Transformed remaining 40u ligation reaction for FNR gfp into 10ul BL21 Transformed 100ng pGFPuv plasmid into 10ul BL21 Poured new LB+amp plates Plated 20, 40, 100ul of transformed bacteria in SOC media on plates esa Quorum Sensing    ☀ Clarice ☀ Colony PCR for RNG and esaR-GFP colonies from master plate RNG (10X) esaGFP (9X) H2O 13.8 45 Buffer 50 18 MgCl2 20 18 dNTPs 20 9 Primer 1 10 9 Primer 2 10 9 Gotaq 2 1.8 ▪ July 20 Invasin + Listerolysin    ☀ Yun Ting ☀ Miniprep of positive colonies from 30h liquid culture. Eluted in 50ul elution buffer and stored at -20. ▶ C= 225.9ng/ul. 260/280=1.86 ▶ D= 297.7ng/ul. 1.87 Anaerobic Promoter    ☀ Yi Han ☀ 1. pGFPuv transformation worked! 2. Single colony for FNR-GFP + 4 more from plating on 19/7 3. Sent FNR-GFP fusion product for sequencing with RP-Biobricks Suffx - result there is no FNR but there is no GFP 4. Colony PCR for FNR gfp (Colonies 1-5, (-) control) Colony PCR (6X) H2O 19.5ul Buffer 10ul dNTP 6ul FP_Biobricks Suffix 6ul RP_Biobricks SUffix 6ul MgCl2 15ul Taq Polymerase 1.5ul ▪ July 21 Invasin + Listerolysin    ☀ Yun Ting ☀ Colony PCR with thermogradient H2O 70ul dNTP 14ul MgCl2 35ul Taq 3.25ul primers (forward + reverse) 14*2 Template DNA 14 C/D-1,2 Col3. C/D-3,4 Col12. D-5,6 Col 9 D-7,8 Col 10 Negative control (no template) Col 8 ▶ Gel ran for 80V, 1h.      D-1,2: VF2, VR.      D-3,4: FP-prefix, RP-suffix.      D-5,6: FP-prefix, FP_M2.      D-7,8: FP-prefix, FP_invF.      C-1,2: VF2, VR.      C-3,4: FP-prefix, RP-suffix. ▶ Results Notes: Too much template DNA (~250ng). Separate the universal primers (to avoid differing extension time). Anaerobic Promoter    ☀ Chi Yan ☀ Ran gel for 20/7 colony pcr for FNR gfp 1kb, 100bp, colonies negative control Anaerobic Promoter    ☀ Yi Han ☀ Redid colony pcr for above as negative control had a positive band - contamination of PCR negative control still has band - reagents contaminated ▪ July 22 Anaerobic Promoter    ☀ Yi Han ☀ Miniprepped colonies 1-5 for FNR-gfp plasmid Did EcoRI/PstI digest Digest for mastermix (5X) Buffer 10ul EcoRI/PstI 2ul H2O 60ul Total 20ul per tube (400ng plasmid in each tube) Ran gel for RE samples, none were positive clones Anaerobic Promoter    ☀ Chi Yan ☀ PCR to test ‘FNR-GFP plasmid’. Colonies 1-5 from 20/7 + negative control 3 sets of primers    1. FP_BB Prefix, RP BB Suffix    2. GFP-F, GFP-R    3. FP_FNRPromoter_BBP, ORP_FNR_Promoter GFP For each reaction (6X) H2O 73.5ul dNTPs 6ul Forward/Reverse Primer 6ul/6ul MgCl2 15ul Taq polymerase 1.5ul Plasmid 1ul Buffer 30ul ▪ July 23 Invasin + Listerolysin    ☀ Yan Ting ☀ RE digest of inv/hyl plasmid with EcoRI and PstI for 2 hours at 37oC. ▶ Lane 1-4: 1kb ladder, 100bp ladder, RE of colony C, RE of colony D. ▶ Size is correct: 4kb main band (inv+hly part) and 2kb (plasmid backbone). These plasmid DNA should have the part. ▪ July 24 Invasin + Listerolysin    ☀ Yun Ting & Yan Ting ☀ Colony PCR, used with temperature gradient to vary annealing temperature. H2O 127.5 5x buffer 50 dNTP 10 MgCl2 25 Taq 2.5 FP + RP 10*2 Template DNA 75ng per rxn 100V for 45min (can run for longer). Lane 1: 1kb ladder; lane 2: 100bp ladder; lane 3-7: FP prefix + RP suffix (5 repeats with increasing annealing temperatures) has 2 bands ~800 bp & 100-200bp (probably non specific bands, will lower # of cycles in future, use higher annealing temp, lower annealing time). Expected size of inv+hly part is 4.1kb. lane 8-9: VF + M2 has a thick band 800-900bp. Expected size is 600+130bp (VF adds ~130bp compared to FP_prefix) .: doesn’t seem to be correct…. lane 10-11: VF + inv F has no bands. Expected is at least 200bp (bcos universal primers). lane 12: VR + inv F seems to have a small band <100bp. Non specific amplification? Expected is 1.5+0.1 kb (from VR). Colony PCR for inv/hly plasmid using Biobricks Prefix and Suffix primers, and Universal primers VF, M2 (Using gradient PCR and different annealing temperature) for 5 samples Ran gel in same order with 1kb, 100bp followed Anaerobic Promoter    ☀ Chi Yan ☀ Ligation of Cut FNRGFP (17/7) with cut GFP vector 1X reaction Buffer 2ul 200ng vector 1.1ul 3:1 256.5ng Water 3.4ul T4 DNA ligase 1ul Total 20ul ▶ Incubation at room temperature for 2h ▶ Transformation into 10ul BL21 competent cells