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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