User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/01

iGEM UNAM-Genomics-Mexico

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September 1st, 2010 1. Run gel to verify purified PCR product of BBa_I20260 with the changed RBS and the double restriction (SpeI-XbaI) of this purified PCR product. This double restriction will not work for our purpose because it eliminates the J23101 promoter + RBS + GFP. I should’ve done the restriction only with XbaI. Lanes: 1) Ladder; 2) Pure PCR product of BBa_I20260 with the changed RBS; 3) BBa_I20260 with the changed RBS double restriction (SpeI-XbaI); 4-8) Claudia's samples. 2. PCR to change RBS into BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), PCR will be done with Platinum Taq Polymerase. I will make 2 reactions with primers RBS-GFP FWD-J23101 REV (Tubes 1-2). PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3). Control: Preffix FWD-Suffix REV (Tube 3). PCR with Platinum Taq DNA polymerase Volume (ul) 10X PCR Buffer minus M -> 5 10mM dNTP mixture -> 1 50mM MgCl2 -> 1.5 Primer mix (10uM each) -> 1 Platinum Taq DNA Pol -> 0.4 Template DNA -> 2 HPLC -> 35.1 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 4 min 3. 72ºC 10 min 4. Hold 4ºC