Pick bacteria from single colonies by touching with a plastic pipet tip (avoid using wood stick or tooth pick), and resuspend in 10 microliters of sterile water in a Eppendorf tube.
Transfer half of the bacteria (5 microliters) into PCR tubes by a pipet, and lyse at 99 C for 5 min on a PCR machine.
Add 100 microliters of LB medium containing antibiotics onto Eppendorf tubes, and move them into 4 C fridge until bacteria colonies are analyzed by PCR .
In the mean time, prepare enough PCR mix for all the tubes, and dispense 20 microliters of PCR mix into PCR tubes containing 5 microliters of lysed bacteria (total volume of PCR reaction in each tube should be 25 microliters).
Run your PCR (total 25 cycles) with your favorite primers to check your insert.
Analyze your PCR products by agarose gel electrophoresis.
Once you identify the positive clones, go back to your fridge, and grow them overnight in a 5 ml culture for miniprep.