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

From OpenWetWare

Jump to: navigation, search
iGEM 2011 Main project page
Previous entry      Next entry

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!!!



Personal tools