IGEM:UNAM/2009/Notebook/IGEM project/2011/06/02

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Ligation of PBBMRCS-5 plasmid with PSB1T3 plasmid and transformation of these plasmids

  • Today I attached the PBBMRCS-5 plasmid with the PSB1T3 plasmid.

PSB1T3 plasmid was amplified by PCR, this plasmid has RFP and PBBMRCS-5 plasmid has a gentamicin resistance.

  • Objective: Built a standar plasmid with RFP and gentamicin resistance.
  • Volume of plasmids
17.5 μl digestion (1)
22. 5 μl digestion (2)
7.5 μl of no-digested plasmid
  • Ligation

1. Put 2.5 μl of DNA in 10 μl of:

Reagent Quantity
H2O 5.5 μl
Buffer 10 X with dNTPs 1 μl
plasmid 2.5 μl
ligase T4 1 μl

Wait for 8 hours.

  • Transformation of the past ligation
  • 1. Add to each tube of competent cells (100 μlEscherichia coli DH5α) 4 μl of the ligation.
Control (no transformed)(1)
Competent cells + ligate plasmid (enzymes)
Competent cells + enzymes (without plasmid)
  • 2. Leave the competent cells in ice for 20 minutes.
  • 3. Hit of heat to 42°C for 1 minute.
  • 4. Leave in ice for 5 minutes.
  • 5. Add to each tube 1 ml of liquid LB without antibiotic.
  • 6. Incubate for 1 hour to 37 °C.
  • 7. Grow in petri dish with solid LB with gentamicin.