User:Jose Fabricio Lopez/Notebook/Logbook/2011/08/01: Difference between revisions

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Enzymes inactivation= 20 min 65°C


==PCR for Kanamycin==
==PCR for Kanamycin==

Revision as of 22:52, 1 August 2011

iGEM 2011 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Objectives

  • Digestion of Plasmid extraction pSB1AK3/BBa_B0015 for Standard Assembly.
  • PCR for Prom+Kanamycin 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.
Reactives 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 Kanamycin

Reactives Tube 1 Tube 2
H2O 31.5 31.5
Buffer Taq Standard 5 5
dNTPs 8mM 1.25 1.25
DNA Paz' PCR Prom+Km 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

==

Status: Success!!!



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