User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/25

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September 25th, 2010
1. Make colony PCR of the ligations:
 * pSB3K3-J23101 + Δ RBS-GFP E0040 (3 colonies, tubes 1-3)
 * pSB3K3-Min. Blue Promoter + Δ RBS-GFP E0040 (1 colony, tube 4)

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.

PCR with Taq DNA polymerase Reactive (ul x sample)
 * Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:
 * 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 4 min
 * 3. 72ºC 10 min
 * 4. Hold 4ºC


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