User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/09

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August 9th, 2010
1. Plasmid extraction to BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040) strain incubated on August 8th.
 * Plasmid extraction was done using the High Pure Plasmid Isolation Kit Roche.

2. Make PCR to change RBS into BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), this because we have one construction with this plasmid + Blue Promoter + GFP E0240, and we want to have both GFP’s under the same RBS, so we can characterize better the promoters without noise caused if we used different Ribosome Binding Sites.
 * PCR will be done with Taq Polymerase.
 * I will use primers RBS_GFP FWD-J23101 REV
 * PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3).
 * Tubes are marked 1-2:
 * 1. BBa_I20260 RBS-GFP FWD-J23101 REV
 * 2. Positive control: BBa_K137019 (~2855 bp) using primers Preffix FWD-Suffix REV.


 * PCR with Taq DNA polymerase
 * Reactive (ul x sample)
 * Taq Polymerase -> 1
 * Taq Reaction Buffer 10X -> 5
 * MgCl 50mM (can be used up to 3ul) -> 2.5
 * dNTP’s 0.4ug/ul -> 2.5
 * Primer Forward (can be used up to 3ul)	-> 2.5
 * Primer Reverse (can be used up to 3ul)	-> 2.5
 * HPLC -> 32
 * DNA -> 2
 * Total volume -> 50


 * Thermocycler program:
 * 1. 95ºC 5 min
 * 2. 35 cycles
 * 95ºC 45 seg
 * 60ºC 45 seg
 * 72ºC 1.5 min
 * 3. 72ºC 5 min
 * 4. Hold 4ºC


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