User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/18

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June 18th, 2010

1. Make transformations with 1) YFP reporter plasmid (BBa_K117008), 2) GFP reporter (E0240), 3) backbone plasmid (pSB3K3), 4) J23101-GFP as reference for promoter measurement (BBa_I20260), 5) p17, 6) p18, 7) Control -.

  • YFP reporter plasmid (BBa_K117008): 2009 Dist. Kit Plate 2, well 14L, plasmid pSB1A2, Amp resistance.
  • GFP reporter (E0240): 2009 Dist. Kit Plate 1, well 12M, plasmid pSB1A2, Amp resitance.
  • Backbone plasmid (pSB3K3-P1010): 2009 Dist. Kit Plate 1, well 7M, Kan resistance.
  • J23101-GFP reference for promoter measurement (BBa_I20260): 2009 Dist. Kit Plate 2, well 17F, plasmid pSB3K3, Kan resistance.
  • Transformation method:
a) Unfreeze competent cells (keep on ice).
b) Add 5ul of exogenous DNA to 100ul of compentent cells.
c) Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
d) Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
e) Incubate 5 min on ice.
f) Transfer the cells to a tube with 1ml of LB medium
g) Incubate the cells in the LB medium for 1hr at 37ºC and shaking (during this period the cells recover their metabolic activity, replicate and synthesize the proteins encoded in the material just inserted).
h) Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
i) Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
j) Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
k) Incubate the plates at 37ºC overnight.

2. PCR to test reactives (dNTP’s and primers).

  • Template is BBa_K137019 (2855 bp).
  • Primers are Preffix FWD and Suffix REV.
  • Samples: (1-5 Taq Polymerase). 1) Old dNTP`s, old Pimers, new buffer and MgCl2; 2) New dNTP’s, new primers, new buffer and MgCl2; 3) Old dNTP`s, new primers, new buffer and MgCl2; 4) New dNTP’s, old primers, new buffer and MgCl2; 5) New dNTP’s, new primers, old buffer and MgCl2; 6) Old dNTP’s, old primers, rTth; 7) New dNTP’s, new primers, rTth; 8) Old dNTP’s, new primers, rTth; 9) New dNTP’s, old primers, rTth.