User:Jose Fabricio Lopez/Notebook/Logbook/2011/08/01

iGEM 2011

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Objectives Digestion of Plasmid extraction pSB1AK3/BBa_B0015 for Standard Assembly. PCR for BBa_P1003 with preffix and suffix primers. See in gel digestion and product of PCR. Clean today´s PCR product. See gel to perform double digestion. Overnight double digestion with EcoRI and SpeI for PCR purified. Digestion Digestion of plasmid pSB1AK3/BBa_B0015 with EcoRI and XbaI. 8 hrs, 37°C. Reagents μL H2O 5 Buffer EcoRI NEB 6 BSA 10X 6 DNA pSB1AK3/BBa_B0015 40 EcoRI NEB 1.5 XbaI NEB 1.5 Enzymes inactivation= 20 min 65°C PCR for BBa_P1003 Reactives Tube 1 Tube 2 H2O 31.5 31.5 Buffer Taq Standard 5 5 dNTPs 8mM 1.25(.2mM) 1.25(.2mM) DNA Paz's PCR BBa_P1003 1 1 Oligo F 5 5 Oligo R 5 5 Taq Standard 1.25 1.25 Total 50 50 Initialization step: 3:00min 95°C Denaturation step: 30seg 94°C Annealing step: 30seg 55°C Extension/elongation step: 40seg 72°C Final elongation 30seg 72°C Final hold: 3hrs 4°C Gel of PCR and digestion 1 Gene'O Mixed Ruler 2 Paz's PCR ~1Kbp as expected 3 Tube 1 PCR ~1Kbp as expected 4 Tube 2 PCR ~1Kbp as expected 5 Dobuble Digestion ~3.4Kbp as expected PCR product purification Roche´s kit. Final dilution= 60μL Gel of PCR purified Vs Plasmid Cut This gel just show concentration between backbone and insert and concentration for Double Digestion of insert(Purified PCR product) Double Digestion of PCR product Double Digestion with EcoRI-HF and SpeI Reagents μL Buffer4 10x 6 BSA 10x 6 PCR product(BBa_P1003) 40 SpeI 1 EcoRI-HF 1 H2O 6 Total 60 Status Success!!!