"
Team:CAU China/J Aug
From 2013.igem.org
 |
 |
 |
 |
 |
|
 |
 |
|
 |
 |
|
AugustWeek 1 August 1st Wet labTransformation of pET-28a(+) for plasmid propagation Digestion of vector with BamHI and SalI PCR for Nadh1 and Sadh2 Gel electrophoresis of digested product and PCR product Plasmid DNA isolation for pET-28a(+) August 2ed Wet labGel purification of digested vector gel product and PCR product Gel check of plasmid extraction Digestion of Sadh2 PCR product with BamHI and SalI Ligation of Sadh2 with pET-28a(+) August 3rd Wet labTransformation of ligated pET-28a(+)-Sadh2 Clone PCR detection for transformation Small inculation of pET-28a(+)-Sadh2(1st) Dry labCalculation of the entropy evolution Week 2 August 4th Wet labDigestion of vector with BamHI and EcoRI Digestion of Nadh1 PCR product with BamHI and EcoRI Gel electrophoresis of digested vector product Gel purification of digested vector gel product Ligation of Nadh1 with pET-28a(+) August 5th Wet labTransformation of ligated pET-28a(+)-Nadh1(1st) Colony PCR identification for transformation
Project interim report Brainstorming sessions for division of work and experiment Dry lab August 7thWet lab Plasmid extraction for pET-28a(+)-Sadh2 Restriction analysis and DNA sequencing of pET-28a(+)-Sadh2 for identification Result:positive! 
Dry lab August 8thWet lab Ligation of Nadh1 with pET-28a(+) Transformation of ligated pET-28a(+)-Nadh1(2nd) Result:no clone(⊙o⊙)! Dry lab August 9thWet lab Ligation of Nadh1 with pET-28a(+) Transformation of ligated pET-28a(+)-Nadh1(2nd) Result:no clone o_O??? Dry lab August 11thWet lab Discussion for experiment PCR of Nadh1 Gel electrophoresis of PCR product Digestion of vector and PCR product with BamHI and EcoRI (for a longer time) Gel electrophoresis of digested vector product Gel purification of digested vector gel product Column- purification of digested gene product Ligation of Nadh1 with pET-28a(+) August 12thWet lab Transformation of ligated pET-28a(+)-Nadh1(3rd) Colony PCR detection for transformation Result:no positive clone (+﹏+)~ Dry lab Calculation of the entropy evolution in another way August 13thWet lab Discussion for experiment Dry lab August 14thWet lab Inoculation of pET-28a(+): repeat Inoculation Dry lab Primers redesign for Nadh1 August 15thWet lab Clean the lab Preparing IPTG for protein induction Autoclave the materials Dry lab August 16thWet lab Small inoculation for pET-28a(+)-Sadh2 Large inoculation for pET-28a(+)-Sadh2 Protein induction with 0.1mM IPTG, 37℃ Cell lysis Enzyme activity assay(1st) Dry lab August 18thWet lab Informal lab meeting August 19thWet lab Small inoculation for pET-28a(+)-Sadh2 Large inoculation for pET-28a(+)-Sadh2 Protein induction with 0.5mM IPTG ,16℃ Cell lysis SDS-PAGE for protein induced assay Result:no protein induced Dry lab August 20thWet lab Small inoculation for pET-28a(+)-Sadh2 Large inoculation for pET-28a(+)-Sadh2 Protein induction with 0.1mM and 0.5mM IPTG,4℃ Cell lysis SDS-PAGE for protein induced assay Result: protein in precipitation not separate well Dry lab August 21stWet lab SDS-PAGE again for induction yesterday Result:no protein induced in precipitation Dry lab August 22ndWet lab New primers for Nadh1 arrival \(^o^)/YES！ PCR for Nadh1 with new primers Gel electrophoresis of PCR product Digestion of vector and PCR product with BamHI and NotI Gel electrophoresis of digested vector product Gel purification of digested vector gel product Column-purification of digested gene product Ligation of Nadh1 with pET-28a(+) Dry lab August 23rdWet lab Transformation of ligated pET-28a(+)-Nadh1(4th) Result:no clone Ligation of Nadh1 with pET-28a(+) Dry lab August 24thWet lab Transformation of ligated pET-28a(+)-Nadh1 (5th) Result:no clone o(︶︿︶)o Dry lab
August 25thLab meeting and discuss for experiment August 26thWet lab Get the ta0841 (commercially synthesized CDS, BGI Crop) PCR for ta0841 amplification Gel electrophoresis of PCR product Dry lab August 27thWet lab Digestion of vector and PCR product with BamHI and SalI Gel electrophoresis of digested vector product Gel purification of digested vector gel product Column-purification of digested gene product Ligation of ta0841 with pET-28a(+) Dry lab August 28thWet lab Transformation of ligated pET-28a(+)-ta0841 (1st) Preparing of LB and autoclave LB-Kanamycin, ampicillin and chloramphenicol plates Order restriction endonuclease(NEB) and DNA Marker Result:no colone Dry lab August 29stWet lab Ligation of ta0841 with pET-28a(+) Ligation of Nadh1 with pET-28a(+) Transformation of ligated pET-28a(+)-ta0841 (2nd) and pET-28a(+)-Nadh1 (6st) Dry lab August 30thWet lab Colony PCR for transformation product identification Small inoculation of pET-28a(+)-ta0841 (2st) and pET-28a(+)-Nadh1 (6st) for restriction anlysis Dry lab August 31stWet lab Plasmid extraction from inoculation yesterday Result:none plasmid isolation Supercompetent cell Preparation for E.coli DH5αand JM109  |
