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.

17.5 μl digestion (1) 22. 5 μl digestion (2) 7.5 μl of no-digested plasmid
 * Volume of plasmids

1. Put 2.5 μl of DNA in 10 μl of:
 * Ligation

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.