User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/13

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August 13th, 2010
1. Repeat 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 Platinum Taq Polymerase.
 * I will use primers RBS-GFP FWD-J23101 REV.
 * PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3).
 * I will use different volumes of DNA (0.1ul, 0.25ul, 0.5ul, 0.75ul, 1ul)
 * Tubes are marked 1-6:
 * 1. BBa_I20260 RBS-GFP FWD-J23101 REV (0.1ul DNA)
 * 2. BBa_I20260 RBS-GFP FWD-J23101 REV (0.25ul DNA)
 * 3. BBa_I20260 RBS-GFP FWD-J23101 REV (0.5ul DNA)
 * 4. BBa_I20260 RBS-GFP FWD-J23101 REV (0.75ul DNA)
 * 5. BBa_I20260 RBS-GFP FWD-J23101 REV (1ul DNA)
 * 6. BBa_I20260 Suffix FWD-J23101 REV (1ul DNA)


 * PCR with Platinum Taq DNA polymerase (ul)
 * 10X PCR Buffer minus M -> 5
 * 10mM dNTP mixture -> 1
 * 50mM MgCl2 -> 2.5
 * Primer mix (10uM each) -> 1
 * Platinum Taq DNA Pol -> 0.4
 * Template DNA -> 2
 * HPLC -> 35.1
 * Total volume -> 50


 * Thermocycler program:
 * 1. 95ºC 5 min
 * 2. 35 cycles
 * 95ºC 45 seg
 * 55ºC 45 seg
 * 72ºC 4 min
 * 3. 72ºC 10 min
 * 4. Hold 4ºC


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