One Step Procedure for Screening Recombinant Plasmids by Size
Inoculate 3.5 mL aliquots of LB media containing the appropriate antibiotic with single colony isolates of the bacteria containing the plasmid in question.
Incubate overnight at 37°C.
Place 150 µL of each culture into a microfuge tube.
Centrifuge in the microfuge for 30 seconds to pellet the bacteria.
Discard the supernatants.
Add 40 µL of agarose beads to each sample.
Add 14 µL of phenol:chloroform (1:1) to each sample and vortex for 5-10 sec.
Centrifuge the samples in the microfuge for 5 min to separate the phases.
Layer 10 µL of each of the aqueous phases onto a 1.2% agarose horizontal gel made up with 1X TPE buffer.
In the outside lanes run lambda DNA digested with HindIII and/or StyI for size markers.
Also run the original vector to show the size of the plasmid without insert DNA.
Gels may be double combed to facilitate running more samples.
Electrophorese the gel in 1X TPE buffer for approximately 400-450 volt-hours.
Stain the gel with ethidium bromide at a concentration of 0.5 µg/mL in d-H2O for 30 minutes.
Observe the gel on a UV light box and photograph the gel.
For further analysis, plasmid DNA may be isolated from those samples that indicate that they have inserts of the appropriate size.
Destain the gel with d-H2O.
