User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/15

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October 15th, 2010

1. Test cells in the microscope for GFP expression.

  • pSB4A5 + MinBP + ΔRBS + GFP E004
  • I20269 (pSB3K3 + J23101 + GFP E0040)


2. Make PCR to test ligation pSB4A5 + MinBP + ΔRBS + GFP E004

  • 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 1-3, 6-10, tubes are marked the same way).
  • Positive control: BBa_I51020.
  • 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
Number of samples -> 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. Make the following ligation:

  • pSB3K3 + J23101 SpeI-PstI (3ul) with ΔRBS + GFP E004 XbaI-PstI (5ul)
  • Ligation methods (Total volume 20ul):
DNA -> Volume of each part to be ligated is indicated above.
Buffer for T4 DNA ligase 5X -> 4ul (Final concentration 20%)
T4 DNA ligase -> 1ul
HPLC -> Complete total volume (20ul)
Incubate overnight at 16ºC
Mix well (vortex) buffer and reaction tubes, unfreeze buffer on ice.



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