Quick Screening Of Colonies For Plasmid With Insert
SP, Dec 05
After transformation of bacteria and overnight growth of antibiotic-resistant colonies, one often needs to identify the colonies containing the DNA of interest (usually, plasmid DNA with an insert). In certain cases where a high success-rate is expected (such as after ligation of a small insert into a vector using sticky-ends created using two different restriction enzymes), only a few colonies can be picked and plasmid DNA prepared from all using Qiagen plasmid mini-prep columns, etc.
However, in cases where a lower efficiency of right ligation is expected (such as in case of blunt-end cloning), one may need to screen many colonies. Kit based methods can become cost or effort-intensive. The following three are alternate and cheap methods in such cases.
Pick up colonies with a 200 ul pipette-tip - the colony will stick - then dispense into tubes containing 50-200 ul culture medium (with the selection agent, antibiotic) and pipet 3-4 times to resuspend the colonies.
Alternately, you can grow picked-up colonies overnight in 2-3 ml culture medium.
Set up PCR reactions (25 ul volume, 25-30 cycles) using 1 ul of bacteria. The primers can be insert-specific or both against insert as well as the vector (e.g., a T7 primer can be used; T7 sites are on many plasmids next to the cloning sites) - in case of latter, one can infer the orientation of the insert.
Electrophorese PCR products.
Restriction enzyme digestion-based screening
Direct lysis buffer
2 mg/ml lysozyme and 0.2 mg/ml RNase A in 15% sucrose (w/v), 10 mM Tris at pH 8, 1mM EDTA and 0.1 mg/ml BSA.
For 10 ml of 1x stock, use 100 ul of 1 M Tris, 200 ul of 0.5 M EDTA, 1.5 g sucrose, 20 mg lysozyme, 100 ul of 20 mg/ml RNase A and 100 ul of 10 mg/ml BSA.
This can be done with either overnight cultures or colony resuspensions described above. Use 1 ml of overnight culture (pellet the bacteria) or as much as possible of the colony resuspension (colonies should be 0.5 mm or larger in size and resuspended in 20 ul; unless the colony is large - like 2 mm - leave the mini-culture suspensions at 37 deg for 2-4 h).
Resuspend in 50 ul of 1x stock (if using overnight culture) or 20 ul of 2x stock (with 20 ul of colony resuspension; remember to leave some for future cultures) of 'direct lysis buffer' and vortex.
Boil for exactly 90 secs.
Spin down at high speed (12k-20k g) at RT for 10 min and collect supernatant.
Use all or 10-20% (in case of overnight cultures) for restriction enzyme digestion, electrophoresing all of the reaction at end.
Plasmid DNA migration-based screening
This is based on the principle that a plasmid containing an insert will be longer in size and thus migrate faster (being more super-coiled) than a plasmid without one. This principle is not applicable to cases where the insert is small relative to the vector (thus not making a discernible difference to the plasmid's migration). A method is described here - Cracking gel method.