User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/05

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October 5th, 2010
1. Make colony PCR of the following strains:
 * pSB1A2 + J23101 (SpeI-PstI restriction) + Δ RBS + GFP E0040: Colonies 1 (tube 1, T1), 3 (T2), 5(T3).
 * Religation of pSB3K3 + J23101 (SpeI restriction): 10 colonies (tubes 4-13)
 * Religation of pSB4A5 (p30) + Minimmum Blue Promoter: Colonies 1-3 (T14-T16), 5 (T17), 7-10 (T18-T21).


 * 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
 * -55ºC 45 seg
 * -72ºC 1 min
 * 3. 72ºC 10 min
 * 4. Hold 4ºC


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