Team:UNICAMP-EMSE Brazil/Notebook/26 August 2011

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31


Electrophoresis Gel 1, 26/08/2011.
Gel 1: Terminator 1 (checking if it is the Terminator vector linearized)/ SoxS C7/8 (d)/ SoxS c7/8 (d)/ SoxS C9 (d)/ RBS+QseB+RBS+Qsec+T C1 (d)/ RBS+QseB+RBS+Qsec+T C2 (d)/ RBS+QseB+RBS+Qsec+T C3 (d)/ RBS+QseB+RBS+Qsec+T C4 (d).

26 August 2011

RBS+QseB+RBS+QseC+T (E-P) and SoxS

Objective:

  • Checking of RBS+QseB+RBS+QseC+T (E-P) and SoxS (linearized with EcoRI) colonies from transformation

Samples: GEL 1

  • Terminator 1 (checking if it is the Terminator vector linearized)/ SoxS C7/8 (d)/ SoxS c7/8 (d)/ SoxS C9 (d)/ RBS+QseB+RBS+Qsec+T C1 (d)/ RBS+QseB+RBS+Qsec+T C2 (d)/ RBS+QseB+RBS+Qsec+T C3 (d)/ RBS+QseB+RBS+Qsec+T C4 (d).
  • Ladder:
  • Bio-Rad 100bp – 10.000bp
  • Gel Agarose concentration:
  • 1%

OBS:

  • D=digested
  • Nd= non-digested


  • Results:

Gene Total size (gene+vector) Linear vector size Gene size Result
Terminator 1 ~3300 pb 3800 pb No cut OK
SoxS C7/8 ~2900 pb 2800 pb No cut OK
SoxS c7/8 ~2900 pb 2800 pb No cut OK
SoxS C9 ~2900 pb 2800 pb No cut OK
RBS+QseB+RBS+Qsec+T C1 4981 pb 2800 pb 2181 pb OK
RBS+QseB+RBS+Qsec+T C2 4981 pb 2800 pb 2181 pb OK
RBS+QseB+RBS+Qsec+T C3 4981 pb 2800 pb 2181 pb OK
RBS+QseB+RBS+Qsec+T C4 4981 pb 2800 pb 2181 pb OK



Samples RBS+QseB+RBS+Qsec+T C1 and SoxS C7/8 were chosen to be digested for further purification and ligation.

Electrophoresis Gel 2, 26/08/2011.
HlyA C1/HlyA C2/ FlhDC /constitutive promotor/ RBS+HlyB/RBS+SoxR+T/RBS+IL-12/ RBS+IL 10/RBS+GFP

Digestion and purification of genes

Objective:

  • Digestion and purification of genes for further ligation reaction.
  • Samples: GEL 2
  • HlyA C1/HlyA C2/ FlhDC /constitutive promotor/ RBS+HlyB/RBS+SoxR+T/RBS+IL-12/ RBS+IL 10/RBS+GFP
  • Ladder:
  • Bio-Rad 100bp – 10.000bp
  • Gel Agarose concentration:
  • 1,5%



  • Results:

Gene Total size (gene+vector) Linear vector size Gene size Result
HlyA C1 3027 pb 2800 pb 277 pb Ok, complete digestion
HlyA C2 3027 pb 2800 pb 227 pb Ok, complete digestion
flHDC promoter ~2926 pb 2926 pb No cut Ok, linearization complete
RBS+HlyB 4983 pb 2800 pb 2183 pb Ok, problably mRFP cut out
RBS+SoxR+Terminator ~3823 pb 3300 pb 618 pb Ok, parcial digestion, no linear vector detect
RBS+IL12 4458 pb 2800 pb 1658 pb Ok, complete digestion
RBS+IL10 3345 pb 2800 pb 545 pb Ok, complete digestion
RBS+GFP 2814 pb 2079 pb 735 pb Ok, complete digestion




Digestions recipes for checking the miniprep:

  • vector+SoxS_promoter (E)
  • 10.5ul - milli-Q watter
  • 2ul - vector+SoxSpromoter
  • 1.5ul - 10X EcoRI Buffer
  • 1ul - EcoRI
  • TOTAL = 15ul
  • RBS+QseB+RBS+QseC+Term (E-P)
  • 14ul - milli-Q watter
  • 2ul - RBS+QseB+RBS+QseC+Term
  • 2ul - 10X Buffer O
  • 1ul - EcoRI
  • 1ul - PstI
  • TOTAL = 20ul


Tasks:

  • Incubate both reactions at 37°C for 1-2 hours
  • Electrophoresis to confirm

Digestions for purification:

  • HlyA (E-S)
  • 32ul - RBS+IL-10
  • 4ul - 10X Buffer R
  • 0.8ul - EcoRI
  • 3.4ul - SpeI
  • TOTAL = 40ul
  • RBS+IL-10 (X-P)
  • 32ul - RBS+IL-10
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+IL-12 (X-P)
  • 32ul - RBS+IL-12
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+SoxR+Term (X-P)
  • 32ul - RBS+SoxR+Term
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+GFP (X-P)
  • 32ul - RBS+GFP
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+HlyB (X-P)
  • 32ul - RBS+HlyB
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • RBS+QseB+RBS+QseC+Term (X-P) (after the miniprep was checked)
  • 32ul - RBS+QseB+RBS+QseC+Term
  • 4ul - 10X Tango Buffer
  • 1.4ul - XbaI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • vector+ConstitutivePromoter (S-P)
  • 32ul - vector+Constitutive_promoter
  • 4ul - 10X Tango Buffer
  • 1.4ul - SpeI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • vector+FlhDC_promoter (S-P)
  • 32ul - vector+FlhDC
  • 4ul - 10X Tango Buffer
  • 1.4ul - SpeI
  • 2.6ul - PstI
  • TOTAL = 40ul
  • vector+SoxS_promoter (S-P) (after the miniprep was checked)
  • 32ul - vector+SoxS
  • 4ul - 10X Tango Buffer
  • 1.4ul - SpeI
  • 2.6ul - PstI
  • TOTAL = 40ul

Tasks:
Incubate reactions at 37°C for 3-4hours.
Electrophoresis (loading the whole digestions volume - join two or more wells in the gel)
Purify the bands
Electrophoresis to confirm (load 5ul of gel purified digestion)

Ligation recipes

  • RBS+HlyD - RBS+TolC+terminator(vector)
  • 6.5ul - milli-Q water
  • 10ul - RBS+HlyD (~300ng)
  • 0.5ul - TolC+terminator(vector) (~150ng)
  • 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






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