Cracking Gel Method To Screen Bacterial Colonies
to identify likely-positive clones after transformation
Santosh Patnaik, Jul 2004
A cracking buffer is used to crack open the bacterium, releasing its DNA (both genomic and plasmid), which is electrophoresed on an agarose gel. The genomic DNA being Mbs in length migrates very slowly while the plasmid DNA move much faster. Plasmid DNA containing insert move slower than those without any; thus possibly positive clones can be identified.
5x Cracking buffer
Sucrose 25 g
sDDW 40 ml
(Dissolve sucrose at 65 deg C)
5 M NaOH 5 ml
10% SDS 2.5 ml
Add sDDW to 50 ml final volume
Take 1 ml aliquot and add 20-40 ul normal 6X or 10X DNA gel loading buffer (or just bromophenol blue; the cracking buffer is dense enough for DNA to fall down into the well) before use. The buffer, with dye, can be kept for less than month (alkaline condition).
Pick single colony with sterile toothpick (as many colonies as necessary to get desired result).
Transfer to 15 ml snap tube containing 2-3 ml LB + antibiotics.
Grow overnight at 37 deg C with 250 rpm shaking
Mix 20 ul overnight culture with 5 ul 5x cracking buffer by pipetting and load onto gel.
Bacterial colonies of about 1mm size can directly be cracked, right after picking from the plate (no overnight culture needed). To do so, pick the colony with a tooth-pick, and resuspend it in 20 ul LB medium or just water (by vigorously rotating and scraping the pick against wall of the tube). Add cracking buffer to that 20 ul of bacterial suspension. Put the pick into a plastic tube with LB and antibiotics for overnight culture (or just inoculate an LB/antibiotic agar plate at specific position).
RNAse A may be added to the cracking buffer (this will decrease the background smear over the lane that arises from bacterial RNA). You can simply use the buffer P1 (containing RNAse) of plasmid preparation kits (5ul cracking buffer with the dye, 15ul bacteria, 5ul P1).
Also see Quick colony screening