Team:CAU China/J Aug

From 2013.igem.org

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August

Week 1 August 1st Wet lab

Transformation 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 lab

Gel 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 lab

Transformation of ligated pET-28a(+)-Sadh2

Clone PCR detection for transformation

Small inculation of pET-28a(+)-Sadh2(1st)

Dry lab

Calculation of the entropy evolution

Week 2 August 4th Wet lab

Digestion 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 lab

Transformation of ligated pET-28a(+)-Nadh1(1st)

Colony PCR identification for transformation


Result:no positive clone

Project interim report

Brainstorming sessions for division of work and experiment

Dry lab

August 7th

Wet lab

Plasmid extraction for pET-28a(+)-Sadh2

Restriction analysis and DNA sequencing of pET-28a(+)-Sadh2 for identification

Result:positive!

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Dry lab

August 8th

Wet lab

Ligation of Nadh1 with pET-28a(+)

Transformation of ligated pET-28a(+)-Nadh1(2nd)

Result:no clone(⊙o⊙)!

Dry lab

August 9th

Wet lab

Ligation of Nadh1 with pET-28a(+)

Transformation of ligated pET-28a(+)-Nadh1(2nd)

Result:no clone o_O???

Dry lab

August 11th

Wet 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 12th

Wet 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 13th

Wet lab

Discussion for experiment

Dry lab

August 14th

Wet lab

Inoculation of pET-28a(+): repeat Inoculation

Dry lab

Primers redesign for Nadh1

August 15th

Wet lab

Clean the lab

Preparing IPTG for protein induction

Autoclave the materials

Dry lab

August 16th

Wet 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 18th

Wet lab

Informal lab meeting

August 19th

Wet 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 20th

Wet 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 21st

Wet lab

SDS-PAGE again for induction yesterday

Result:no protein induced in precipitation

Dry lab

August 22nd

Wet 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 23rd

Wet lab

Transformation of ligated pET-28a(+)-Nadh1(4th)

Result:no clone

Ligation of Nadh1 with pET-28a(+)

Dry lab

August 24th

Wet lab

Transformation of ligated pET-28a(+)-Nadh1 (5th)

Result:no clone o(︶︿︶)o

Dry lab


August 25th

Lab meeting and discuss for experiment

August 26th

Wet lab

Get the ta0841 (commercially synthesized CDS, BGI Crop)

PCR for ta0841 amplification

Gel electrophoresis of PCR product

Dry lab

August 27th

Wet 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 28th

Wet 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 29st

Wet 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 30th

Wet 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 31st

Wet lab

Plasmid extraction from inoculation yesterday

Result:none plasmid isolation

Supercompetent cell Preparation for E.coli DH5αand JM109

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