User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/25

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October 25, 2010

1. Extract plasmid from colonies 1-10 incubated on October 24, the plasmid should contain: pSB3K3 + J23101 + ΔRBS + GFP E004

  • Plamid extraction was made using the QIAprep Spin Miniprep Kit.


2. Made PCR to test pSB3K3 + J23101 + ΔRBS + GFP E004 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 1-10, 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 1:00 min
3. 72ºC 10 min
4. Hold 4ºC


3. Run gel to verify PCR’s made on October 23 and 25.

Gel 25Oct2010.JPG

Lanes: 1) Green ladder; 2) MinBP + ΔRBS + GFP E004 Colony 1 (C1) (expressing RFP); 3) MinBP + ΔRBS + GFP E004 (C7); 4) BBa_I51020 Ctrl + 23 Oct.; 5-14) pSB3K3 + J23101 + ΔRBS + GFP E004 (Colonies 1-10, respectively); 15) BBa_I51020 Ctrl + 25 Oct.


4. Incubate overnight in M9 minimum medium suplmented with glycerol (0.4%) the following constructions:

  • MinBP + ΔRBS + GFP E004 in pSB1C3 (37º and 25º C, in the dark, colonies 7 & X)
  • J23101 + ΔRBS + GFP E004 in pSB3K3 (37ºC, colonies 3 & 5)
  • MinBP in pSB4A5 (37ºC and 25ºC, in the dark, colonies 3 & 5)