@It is very often most convenient to screen clones for the presence of some sequence as colonies, as opposed to isolating plasmid DNA & screening the DNA from Southerns. Although not as "clean" as Southerns, colony lifts are nonetheless a standard lab procedure for screening clones.
@Although it is possible to lift colonies directly from plated-out transformation reactions, it is best to "grid-out" the colonies first so that extraneous colonies (i.e. blue colonies) are eliminated,allowing more legitimate colonies per plate screened and to make it easier to identify which colony on the plate is giving a hybridization signal. If the gridding is done in duplicate, it also gives you a source of each clone that hasn't been contaminated by the nitrocellulose filter.
- Reagents
- 1% SDS/5mM EDTA
- 2X SSC - diluted 1:10 from 20X SSC
- Lysis solution
- Neutralization solution
- 20X SSC
|
final |
per liter |
| NaCl |
3M |
175.3 grams |
| Na citrate |
0.3M |
88.2 grams |
Adjust to pH 7 with 10N NaOH
- Methods
- Spread out some saran-wrap & place 4 3MM squares (large enough for 3 85mm nitrocellulose circles). Pour 1%SDS/5mM EDTA on the first 3MM square, lysis sol'n on the next, neutralization sol'n on the next & 2X SSC on the last. Pour enough solution onto each to saturate the 3MM paper, without leaving pools of liquid on top on the paper.
- Place a DRY nitrocellulose filter (85mm circle) onto each plate to be lifted & tap down enough so that the filter wets all over. Leave 5 min.
- Lift the nitrocellulose filters (most of each colony should adhere to the filter) & place colony-side up onto the 1% SDS/5mM EDTA 3MM paper. Incubate 5 min.
- Transfer the filters to the lysis sol'n 3MM paper & incubate 5 min.
- Transfer the filters to the neutralization sol'n 3MM paper & incubate 5 min.
- Transfer the filters to the 2X SSC 3MM paper & incubate 5 min.
- Place the filters colony-side up onto dry 3MM paper & air-dry 15 min., then bake in a vacuum oven for 2 hr at 80C.
- The filters are now ready for hybridization as usual.