User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/03

iGEM UNAM-Genomics-Mexico

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Augus3rd, 2010 1. Run gel to verify extracted plasmids, PstI and EcoRI-PstI restrictions made on July 7th. Lanes: 1) Ladder; 2) Plasmid L1; 3) Plasmid L2; 4) Plasmid L4, 5) Plasmid L5; 6) Plasmid L6; 7) p18; 8) p19; 9) p30; 10) L1 PstI; 11) L2 PstI; 12) L4 PstI; 13) L5 PstI; 14) L6 PstI; 15) Ladder. Lanes: 1) Ladder; 2) p18 PstI; 3) p19 PstI; 4) p30 PstI; 5) L1 EcoRI-PstI; 6) L2 EcoRI-PstI; 7) L4 EcoRI-PstI; 8) L5 EcoRI-PstI; 9) L6 EcoRI-PstI; 10) p18 EcoRI-PstI; 11) p19 EcoRI-PstI; 12) p30 EcoRI-PstI; 13) Ladder. 2. Make PCR to test constructions: L2: p30-MinBP + GFP BBa_K145015 (74 min); L4: Backbone plasmid pSB3K3 + Blue Promoter BBa_K238013 + GFP BBa_K145015 (74 min). PCR will be done with Taq polymerase, and with primers Preff FWD-Suff REV and BluePromoter FWD-BluePromoter REV. Tubes are marked 1-5 as follows: L2 Preff FWD-Suff REV L4 Preff FWD-Suff REV L2 BluePromoter FWD-BluePromoter REV L4 BluePromoter FWD-BluePromoter REV Positive control using L1 with Preff FWD-Suff 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