User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/30

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October 1st, 2010
1. Make colony PCR with the transformations replated on September 29th:
 * pSB1A2 + J23101 (SpeI-PstI restriction) + Δ RBS + GFP E0040: Colonies 1(tube 1, T1), 3 (T2), 5(T3) and 6 (T4), the later is expressing RFP.
 * Religation of pSB3K3 + J23101 (SpeI restriction): 10 colonies (tubes 5-14)
 * Religation of pSB4A5 (p30) + Minimmum Blue Promoter: Colonies 1-3 (T15-T17), 5 (T18), 7-10 (T19-T22).


 * Colony PCR methods:
 * 1) Take a colony and resuspend in 200ul of Tri-EDTA 10/1-NaCl 10 mM.
 * 2) Heat 10 min at 95ºC.
 * 3) Centrifugue at 14000 rpm 2 min.
 * 4) Take 10 ul as template for PCR.


 * Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:


 * 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 -> 24
 * DNA -> 10
 * Total volume -> 50


 * Thermocycler program:
 * 1. 95ºC 5 min
 * 2. 35 cycles
 * -95ºC 45 seg
 * -60ºC 45 seg
 * -72ºC 1.5 min
 * 3. 72ºC 10 min
 * 4. Hold 4ºC

2. Run gel to verify colony PCR.



Lanes: 1) Green ladder; 2) T2; 3) T1; 4-14) T3-T13; 15) Green ladder.



Lanes: 1) Green ladder; 2-10) T14-T22; 11) Green ladder.


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