User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/23

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October 23, 2010

1. Extract plasmid from colonies 1 and 7 incubated on October 22, the plasmid should contain: MinBP + ΔRBS + GFP E004 in pSB1C3.

  • All colonies except 7 are expressing RFP, that means they are religations of the plasmid without the construction MinBP + ΔRBS + GFP E004.
  • Plamid extraction was made using the QIAprep Spin Miniprep Kit.

2. Made PCR to test MinBP + ΔRBS + GFP E004 in pSB1C3 construction.

  • PCR will be done with Platinum Taq Polymerase.
  • Primers used: Preffix FWD-Suffix REV.
  • Template for the reaction is extracted plasmid from the transformation with this construction (Colonies 1 and 7, tubes are marked the same way).
  • Positive control: BBa_I51020 (p19).
  • PCR with Platinum Taq DNA polymerase -> Volume (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 2.5
Primer mix (10uM each) -> 2
Platinum Taq DNA Pol -> 0.4
Template DNA -> 1
HPLC -> 38.1
Total volume -> 50
  • If primers are separated and in concentration 5uM, use 1ul of each one.
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles:
-95ºC 45 seg
-55ºC 45 seg
-72ºC 1:10 min
3. 72ºC 10 min
4. Hold 4ºC

3. Inactivate ligation enzyme for 20 min at 65ºC.

4. Transform the following ligations:

  • pSB3K3 + J23101 + ΔRBS + GFP E004 (10ul)
  • CBG in pLRE (Mariana’s, 5ul)

into DH5-α competent cells, transform also BBa_I51020 (p19) (5 ul DNA) as control.

  • Transformation method:
  1. Unfreeze competent cells (keep on ice).
  2. Add exogenous DNA to 50ul of compentent cells.
  3. Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
  4. Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
  5. Incubate 5 min on ice.
  6. Transfer the cells to a tube with 1ml of LB medium
  7. 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).
  8. Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
  9. Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
  10. Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
  11. Incubate the plates at 37ºC overnight.