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

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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


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