Difference between revisions of "Team:IIT Kanpur/Notebook"
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| + | </head> | ||
| + | |||
| + | <body lang=EN-IN> | ||
| + | |||
| + | <div class=WordSection1> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>Design construct for secretion assay : | ||
| + | Through July</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>August 28, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of all J. Anderson | ||
| + | promoter</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of received | ||
| + | constructs with XbaI+SpeI for cloning purpose.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>August 30, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Mini prep of J23100, J23114, | ||
| + | J23102, J23110 (J. Anderson’s promoter)</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>August 31, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion (With XbaI+SpeI) of </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt;text-indent:36.0pt'><span lang=EN>pelB | ||
| + | sfGFP</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt;text-indent:36.0pt'><span lang=EN>pelB | ||
| + | linker sfGFP</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt;text-indent:36.0pt'><span lang=EN>ompT | ||
| + | sfGFP</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt;text-indent:36.0pt'><span lang=EN>ompT | ||
| + | linker sfGFP</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> 2. RE Digestion of Kanamycin backbone | ||
| + | with with XbaI+SpeI</span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> 3. Transformation of JA promoter.</span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 2, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of Plasmid Backbone </span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ligation of blunt ended Kanamycin | ||
| + | backbone of received constructs.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 4, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of J23105 </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 7, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Phosphorylation of 5’ ends of</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> pelB-linker-sfGFP</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> ompT-linker-sfGFP</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 8, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ligation of phosphorylated | ||
| + | constructs with Kanamycin backbone. </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 9, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of ligation | ||
| + | performed previous day</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Culture of JA23105, JA23102, | ||
| + | JA23114, JA23110</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 10, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Mini prep of JA23105, JA23102, | ||
| + | JA23114, JA23110</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 12, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Phosphorylation of | ||
| + | pelB-linker-sfGFP and ompT-linker-sfGFP (with changed buffer and new | ||
| + | enzymes)</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 13, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ligation of phosphorylated | ||
| + | constructs with Kanamycin backbone</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 18, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Mini prep of ompT-linker colonies. | ||
| + | (Picked from previous ligation) </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 20, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of ompT-linker with | ||
| + | SpeI to check the enzyme.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 21, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of SDSa backbone | ||
| + | from plasmids gifted by team TecCem</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE digestion repeated (as done on | ||
| + | previous day, with new enzymes)</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 25, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE digestion of JA100 pelB linker | ||
| + | sfGFP with EcorI and PstI. </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 26, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ligation reaction of digested | ||
| + | construct in Chloramphenicol resistance containing backbone</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 27, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <p class=MsoNormalCxSpMiddle style='margin-left:72.0pt;text-indent:-18.0pt'><span | ||
| + | lang=EN>1.<span style='font:7.0pt "Times New Roman"'> </span></span><span | ||
| + | lang=EN>Ligation reaction repeated.</span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 28, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <p class=MsoNormalCxSpMiddle style='margin-left:72.0pt;text-indent:-18.0pt'><span | ||
| + | lang=EN>1.<span style='font:7.0pt "Times New Roman"'> </span></span><span | ||
| + | lang=EN>Ligation reaction with changed parameters for the same insert and | ||
| + | vector as on previous day.</span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:72.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>September 29, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of both ligations | ||
| + | done on previous day</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of plasmid | ||
| + | containing SDSa and plasmid containing JA102 promoter</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE digestion of received construct | ||
| + | with EcorI and PstI</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 1, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of pSBIK3.m1, pSB1A3 | ||
| + | and received constructs from IDT with EcorI and PstI</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ligation reaction of IDT | ||
| + | constructs in pSBIK3.m1and pSB1A3</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 2, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation for the above | ||
| + | ligation. </span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion JA102 backbone with | ||
| + | EcorI and ligating the linearized template to check whether ligase is | ||
| + | working.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 4, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of IDT constructs | ||
| + | with EcorI and PstI</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of template pSBIK3 | ||
| + | and pSB1C3 with EcorI and PstI</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of SDSa</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Culture of SDSa colonies</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ligation of digested templates. </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 6, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of all constructs | ||
| + | in Chloramphenicol and Kanamycin backbones</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 7, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Culture set for SDSa transformed | ||
| + | colonies and experiment for quantitating protein secretion from our cloned | ||
| + | constructs.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>RE Digestion of backbone </span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 8, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Promoter sfGFP analysis of | ||
| + | JA102-pelB-ompT</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>LB chloramphenicol plates were | ||
| + | made</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of JA100, JA102, | ||
| + | JA105, JA108-pelB and JA102, JA105-ompT containing IDT constructs.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>SDSa amplification using PCR</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Culture of constructs for shipping</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Pancau Staining for checking | ||
| + | protein secretion via JA102 pelB/ompT containing backbones</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 9, 2018</span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>PCR amplification of SDSa gene.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Mini prep of JA102 pelB, | ||
| + | JA105-pelB/ompT containing construct</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Interlab experiment doe | ||
| + | quantitating protein secretion performed for JA102 pelB/ompT , </span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Culture set for other quantitative | ||
| + | experiments</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Western blot for JA102 pelB/ompT</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 10, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN> </span></b></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>All parts were shipped.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Anti GFP staining for | ||
| + | JA102/JA105/JA100 pelB, JA102/105 ompT</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Fluorescence and OD measurement at | ||
| + | different time points for above constructs.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Experiment for effects of SDS on | ||
| + | growth of E.coli(DH5 alpha)</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 11, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Fluorescence and OD measurement at | ||
| + | different time points for above constructs.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Western Blot repeated with | ||
| + | supernatant only.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN>October 13, 2018</span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Silver staining of | ||
| + | JA102/JA105/JA100 pelB, JA102/105 ompT</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Culture set for western blot via | ||
| + | anti HIS antibody.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 15, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Western Blot staining with HIS | ||
| + | antibody.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>50ml culture of JA105 pelB | ||
| + | containing secretion assay construct.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 16, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Transformation of all cloned | ||
| + | construct in Rosetta strain or E.coli (BL21)</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Western blot using anti HIS | ||
| + | antibody</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:36.0pt'><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 17, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Set culture for colonies obtained.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <p class=MsoNormal><b><span lang=EN>October 18, 2018</span></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | <ol style='margin-top:0cm' start=1 type=1> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Ponceau staining of the | ||
| + | constructs.</span></li> | ||
| + | <li class=MsoNormalCxSpMiddle><span lang=EN>Western blot using anti HIS | ||
| + | antibody.</span></li> | ||
| + | </ol> | ||
| + | |||
| + | <p class=MsoNormal><span lang=EN> </span></p> | ||
| + | |||
| + | </div> | ||
| + | |||
| + | </body> | ||
| + | |||
| + | </html> | ||
| + | |||
{{Template:IIT_Kanpur/Footer}} | {{Template:IIT_Kanpur/Footer}} | ||
Revision as of 14:35, 17 October 2018
Design construct for secretion assay : Through July
August 28, 2018
- Transformation of all J. Anderson promoter
- RE Digestion of received constructs with XbaI+SpeI for cloning purpose.
August 30, 2018
- Mini prep of J23100, J23114, J23102, J23110 (J. Anderson’s promoter)
August 31, 2018
- RE Digestion (With XbaI+SpeI) of
pelB sfGFP
pelB linker sfGFP
ompT sfGFP
ompT linker sfGFP
2. RE Digestion of Kanamycin backbone with with XbaI+SpeI
3. Transformation of JA promoter.
September 2, 2018
- RE Digestion of Plasmid Backbone
- Ligation of blunt ended Kanamycin backbone of received constructs.
September 4, 2018
- Transformation of J23105
September 7, 2018
- Phosphorylation of 5’ ends of
pelB-linker-sfGFP
ompT-linker-sfGFP
September 8, 2018
- Ligation of phosphorylated constructs with Kanamycin backbone.
September 9, 2018
- Transformation of ligation performed previous day
- Culture of JA23105, JA23102, JA23114, JA23110
September 10, 2018
- Mini prep of JA23105, JA23102, JA23114, JA23110
September 12, 2018
- Phosphorylation of pelB-linker-sfGFP and ompT-linker-sfGFP (with changed buffer and new enzymes)
September 13, 2018
- Ligation of phosphorylated constructs with Kanamycin backbone
September 18, 2018
- Mini prep of ompT-linker colonies. (Picked from previous ligation)
September 20, 2018
- RE Digestion of ompT-linker with SpeI to check the enzyme.
September 21, 2018
- Transformation of SDSa backbone from plasmids gifted by team TecCem
- RE digestion repeated (as done on previous day, with new enzymes)
September 25, 2018
- RE digestion of JA100 pelB linker sfGFP with EcorI and PstI.
September 26, 2018
- Ligation reaction of digested construct in Chloramphenicol resistance containing backbone
September 27, 2018
1. Ligation reaction repeated.
September 28, 2018
1. Ligation reaction with changed parameters for the same insert and vector as on previous day.
September 29, 2018
- Transformation of both ligations done on previous day
- Transformation of plasmid containing SDSa and plasmid containing JA102 promoter
- RE digestion of received construct with EcorI and PstI
October 1, 2018
- RE Digestion of pSBIK3.m1, pSB1A3 and received constructs from IDT with EcorI and PstI
- Ligation reaction of IDT constructs in pSBIK3.m1and pSB1A3
October 2, 2018
- Transformation for the above ligation.
- RE Digestion JA102 backbone with EcorI and ligating the linearized template to check whether ligase is working.
October 4, 2018
- RE Digestion of IDT constructs with EcorI and PstI
- RE Digestion of template pSBIK3 and pSB1C3 with EcorI and PstI
- Transformation of SDSa
- Culture of SDSa colonies
- Ligation of digested templates.
October 6, 2018
- Transformation of all constructs in Chloramphenicol and Kanamycin backbones
October 7, 2018
- Culture set for SDSa transformed colonies and experiment for quantitating protein secretion from our cloned constructs.
- RE Digestion of backbone
October 8, 2018
- Promoter sfGFP analysis of JA102-pelB-ompT
- LB chloramphenicol plates were made
- Transformation of JA100, JA102, JA105, JA108-pelB and JA102, JA105-ompT containing IDT constructs.
- SDSa amplification using PCR
- Culture of constructs for shipping
- Pancau Staining for checking protein secretion via JA102 pelB/ompT containing backbones
October 9, 2018
- PCR amplification of SDSa gene.
- Mini prep of JA102 pelB, JA105-pelB/ompT containing construct
- Interlab experiment doe quantitating protein secretion performed for JA102 pelB/ompT ,
- Culture set for other quantitative experiments
- Western blot for JA102 pelB/ompT
October 10, 2018
- All parts were shipped.
- Anti GFP staining for JA102/JA105/JA100 pelB, JA102/105 ompT
- Fluorescence and OD measurement at different time points for above constructs.
- Experiment for effects of SDS on growth of E.coli(DH5 alpha)
October 11, 2018
- Fluorescence and OD measurement at different time points for above constructs.
- Western Blot repeated with supernatant only.
October 13, 2018
- Silver staining of JA102/JA105/JA100 pelB, JA102/105 ompT
- Culture set for western blot via anti HIS antibody.
October 15, 2018
- Western Blot staining with HIS antibody.
- 50ml culture of JA105 pelB containing secretion assay construct.
October 16, 2018
- Transformation of all cloned construct in Rosetta strain or E.coli (BL21)
- Western blot using anti HIS antibody
October 17, 2018
- Set culture for colonies obtained.
October 18, 2018
- Ponceau staining of the constructs.
- Western blot using anti HIS antibody.
