IGEM:VGEM/2007/Notebook/2007-6-19

June 19, 2007
Getting the DNA out of the Biobrick wells:
 * George- paper presentation
 * Emre - paper presentation
 * Amy and Kevin: Selecting biobrick for today's transformation (BBa_I13521, Ptet mRFP. switch off by tetracycline)
 * Emre, George, Ranjan: Preparing for meeting tomorrow with Morgan Estabrook
 * Transformation Protocol of E.Coli with RFP

1. Puncture a hole through the foil with a pipette tip into the well that corresponds to Part BBA I13521: Ptet mRFP. switch off by tetracycline. 2. Add 15 uL of diH2O (deionized water) to the well

Transforming Cells:

1. Thaw ~50 μl cells on ice. Do not use glass tubes, which adsorb DNA. 1. Thaw another 50 μl of cells for the control plates (these will not be transformed) 2. Add 1 ul DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume) 3. Incubate all cells on ice for 30 minutes. 1. Note: If you are in a rush, you can shorten this incubation time to 5-10 min. 4. Incubate cells for 50 seconds at 42°C. 5. Incubate cells on ice for 2 min.  6. Add 1mL of LB broth to a tube and add all of the cells into it   7. Incubate for 1 hour at 37°C on shaker. 1. Note: Can also save some time here by reducing incubation to ~45 min.        2. Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics 8. Sterilize the spreader 9. Spread 20 & 200 μl of transformed cells and control cells onto plates made with appropriate antibiotic (i.e. 100mg/ml of Amp), four plates in total. 10. Grow overnight at 37°C.