Team:UNICAMP-EMSE Brazil/Notebook/27 August 2011

27 August 2011

Purification of SoxS and RBS+QseB+RBS+Qsec+T and Quantification of 26/Aug/2011 samples:

Electrophoresis Gel 1, 27/08/2011.
Gel 1: SoxS (S/P) / SoxS (S/P)/ SoxS (S/P)/ RBS+QseB+RBS+Qsec+T (X/P)/ RBS+QseB+RBS+Qsec+T (X/P)/ RBS+QseB+RBS+Qsec+T (X/P)/ HlyA C2/ RBS+SoxR+T/RBS+IL 12/ RBS+HlyB/ RBS+GFP/HlyA C1/ RBS+IL 10/fLHDC/Constitutive promoter

Objective:

• Purification of digested SoxS and RBS+QseB+RBS+Qsec+T and quantification of the purification performed yesterday of digested samples.

Samples: GEL 1

• SoxS (S/P) / SoxS (S/P)/ SoxS (S/P)/ RBS+QseB+RBS+Qsec+T (X/P)/ RBS+QseB+RBS+Qsec+T (X/P)/ RBS+QseB+RBS+Qsec+T (X/P)/ HlyA C2/ RBS+SoxR+T/RBS+IL 12/ RBS+HlyB/ RBS+GFP/HlyA C1/ RBS+IL 10/fLHDC/Constitutive promoter.

• Ladder:

• Bio-Rad 100bp – 10.000bp

• Gel Agarose concentration:

• 1%

OBS:

• Results:

Electrophoresis Gel 2, 27/08/2011.
SoxS / RBS+QseB+RBS+Qsec+T

• Only HlyA was not detected by the gel. Problems with this molecule purification from the gel.

Quantification:

Objective:

• Quantification of digested SoxS and RBS+QseB+RBS+Qsec+T purified from the former gel.

• Samples: GEL 2

• SoxS / RBS+QseB+RBS+Qsec+T

• Ladder:

• Bio-Rad 100bp – 10.000bp

• Gel Agarose concentration:

• 1,5%

• Results:

Good bands! Lets ligate them following the schema below:

Digestions recipes

• RBS+IL-10 - SoxS_promoter(vector)

• 9 ul - milli-Q water
• 7 ul - RBS+IL-10 (~60ng)
• 1 ul - SoxS_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

• RBS+GFP - SoxS_promoter(vector)

• 6 ul - milli-Q water
• 10 ul - RBS+GFP (~60ng)
• 1 ul - SoxS_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

• RBS+HlyB - SoxS_promoter(vector)

• 14 ul - milli-Q water
• 2 ul - RBS+HlyB (~50ng)
• 1 ul - SoxS_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

• RBS+QseB+RBS+QseC+Term - Constitutive_promoter(vector)

• 11 ul - milli-Q water
• 1 ul - RBS+QseB+RBS+QseC+Term (~50ng)
• 5 ul - Constitutive_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

After all digestions (from 26/08/2011): lIGATIONS

• HlyA - terminator(vector)

• 3 ul - milli-Q water
• 30.5 ul - HlyA (?ng)
• 1ul - terminator(vector) (~30ng)
• 4ul - 10X T4 Buffer
• 1.5ul - T4 DNA Ligase
• TOTAL = 40ul

• RBS+IL-12 - flhDC_promoter(vector)

• 11.5 ul - milli-Q water
• 4 ul - RBS+IL-12 (~60ng)
• 1.5 ul - flhDC_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

• RBS+SoxR+Term - Constitutive_promoter(vector)

• 8.5 ul - milli-Q water
• 3.5 ul - RBS+SoxR+Term (~60ng)
• 5 ul - Constitutive_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

• RBS+GFP - flhDC_promoter(vector)

• 5.5 ul - milli-Q water
• 10 ul - RBS+GFP (~60ng)
• 1.5 ul - flhDC_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

• RBS+HlyB - flhDC_promoter(vector)

• 13.5 ul - milli-Q water
• 2 ul - RBS+HlyB (~50ng)
• 1.5 ul - flhDC_promoter(vector) (~20ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

Tasks:
Incubate all ligation reactions for 1-2 hour at 22°C. Use 5ul of these ligations to do the transformation (except for HlyA - 20ul)