Team:UNICAMP-EMSE Brazil/Notebook/23 August 2011

Electrophoresis Gel 1, 23/08/2011.
Gel 1:: Ladder / HlyD digested (three joint wells) / HlyD non-digested / HlyA digested (three joint wells) / HlyA non-digested .

23 August 2011

Purification of digested HlyA and RBS+HlyD - secretion system

Objective: Electrophoresis for the purification of digested HlyA and RBS+HlyD.

• Ladder: Bio-Rad 100bp – 10.000bp
• Agarose concentration: 1%
• Samples:

• Ladder / HlyD digested (three joint wells) / HlyD non-digested / HlyA digested (three joint wells) / HlyA non-digested

• Results:

-All the genes were purified using Fermentas kit.

Transformation

The plasmids purified yesterday were transformed using the Chemically Competent E. coli and the protocol of Chemo-transformation.

• Three plates for each Construct were used, inoculated with different volumes of the transformation:

• 100 uL, 200 uL and the remaining volume after centrifugation and concentration of bacteria for the plasmid containing:

• RBS+QseB+QseC+Terminator
• RBS+TolC+Terminator
• RBS+SoxR+Terminator

New digestion for our synthetic promoters and Electrophoresis in a more concentrated gel

Electrophoresis Gel 2, 23/08/2011.

Objective: Check for problems with our synthesized promoters

• Agarose concentration: 1.5% stained with ethidium bromide
• Samples:

• L = 1Kb Plus DNA Ladder (Invitrogen)
• 1 = vector containing the SoxS promoter digested with EcoRI
• 2 = vector containing the SoxS promoter digested with PstI
• 3 = vector containing the SoxS promoter digested with EcoRI and PstI

• Explanation of results:

• Both photos are from the same gel, but the right one is in grey scale. The single digestions (conducted with only one enzyme - lanes 1 and 2) showed that we have both restriction sites in the vector, which is actually present in our synthesized insert because the vector does not have any restriction site. So, there is no mutations here. Lane three shows our released promoter (black arrow), although it is not easy to see.
• So, our promoters are probably correctly inserted in the vectors, with the problem being that they are too short to be visualized after staining with ethidium bromide. Since we do not need to take them off the vector to work with them (we can put our larger fragments in the vector with the promoters), as soon as we have the gene(s)+terminator constructions done we can start putting them in the linearized vectors.

Digestions recipe

• HlyA:

• 50ul - HlyA miniprep
• 8ul - milli-Q water
• 7ul - 10X Buffer R
• 1ul - EcoRI
• 4ul - SpeI
• Total = 70ul

• RBS+HlyD:

• 32ul - RBS+HlyD miniprep
• 4ul - 10X Buffer R
• 0.8ul - EcoRI
• 3.2ul - SpeI
• Total = 40ul

Ligations recipe

• RBS+TolC - vector+terminator

• 9ul milli-Q water
• 5ul - RBS+TolC (~300ng)
• 3ul - vector+terminator (~100ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• Total = 20ul

• vector+terminator - RBS+SoxR

• 3ul - milli-Q water
• 20ul - RBS+SoxR (~80ng)
• 2.5ul - vector+terminator (~80ng)
• 3ul - 10X T4 Buffer
• 1.5ul - T4 DNA Ligase
• TOTAL = 30ul

• RBS+QseB+RBS+QseC - vector+terminator

• 4ul - milli-Q water
• 10ul - RBS+QseB+RBS+QseC (~200ng)
• 3ul - vector+terminator (~100ng)
• 2ul - 10X T4 Buffer
• 1ul - T4 DNA Ligase
• TOTAL = 20ul

Incubate all ligation reactions for 1hour at 22°C. Use 1-2ul of these ligations to do the transformation in the afternoon.

Next Tasks:

1. Incubate both reactions at 37°C for 3-4hours.
2. Electrophoresis (loading the whole digestions volume)
3. Purify the bands
4. Electrophoresis to confirm