Transfer five microliters of electroporated overnight growth L rotary culture from a 96 well plate to a PCR tube. Spin down the cells and discard the supernatant before resuspending the pellet in 20 microliters of 20 millimolar sodium hydroxide. Boil the samples at 95 degrees Celsius for five minutes.
Vortex, and repeat the boiling once before placing the samples on ice. Spin down the cells then take one microliter of the supra natant and dilute into 99 microliters of ice cold DNAs and RNAs free double distilled water. Use the 100 times dilution as the template DNA in a standard PCR reaction, using plasmid specific primers and include a positive control with a plasmid derived from the E coli mini prep.
After PCR, place the samples on ice and add an appropriate loading dye at one X concentration. Run the samples in 1%Agorase gell in TAE buffer at 110 volts for 30 minutes. Colony PCR of transformed L Rotary varying the preparation temperature resulted in differing rates of PCR success.
Samples prepared on ice showed a higher percentage of success rate than that prepared at room temperature or prepared at room temperature, and then incubated at 37 degrees Celsius for 30 minutes before PCR.
