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

iGEM UNAM-Genomics-Mexico

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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: Take a colony and resuspend in 200ul of Tri-EDTA 10/1-NaCl 10 mM. Heat 10 min at 95ºC. Centrifugue at 14000 rpm 2 min. 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