In situ screening of bacterial colonies - Protocols
Patrick Eldin, Faculté de Pharmacie, Montpellier, France

1. Colonies are plated on LB Agar medium (petri dishes) with the correct antibiotic.    

2. Following an overnight incubation at 37°C individual colonies are picked (with a tooth pick) and inoculated into single wells of a Nunc MicroWell™ Plate 96F (cat. no. 269787) prefilled with LB-medium containing the appropriate antibiotic.   

3. Following an overnight incubation at 37°C a disposable TSP 96 Pin Replicator (cat. no. 445497) is used to replicate all individual colonies from one plate to a nylon hybridization filter (e.g. Pall Biodyne™ B Nunc Nylon Membrane (cat. no. 250305)) (fig. 1). Allow filter to air-dry. At this stage, add one tenth volume of glycerol to each well of the 96 MicroWell plate overnight culture to allow the storage of the colonies at –80°C.   

4. The air-dried nylon filter is then placed (colonies facing up) on a Nunc OmniTray (cat. no. 242811) previously filled with LB Agar containing appropriate antibiotic. Incubate overnight 37°C to allow the cells to grow on the filter.

Fig. 1
By using the disposable TSP-96 Pin Replicator (cat. no. 445497) all individual colonies of a plate are replicated on a nylon hybridization filter (e.g. Pall Biodyne™ B Nunc Nylon Membrane (cat.no. 250305)).


B. Bacterial Lysis on filter

1. The alkaline lysis is performed as follows:
– 1.0 ml of the lysis solution (0.5 N NaOH) is placed in an OmniTray. The filter is placed on top of the lysis solution with the colony side up. Incubate at room temperature for 10 minutes. The solution diffuses through the filter and lyses the cells during the incubation.
  – The same incubation is repeated with a fresh lysis solution.   

2. At the end of the second 10 minutes incubation the filter is neutralized using 1 ml of Tris 1M pH 5.4.Repeat this treatment twice.   

3. The filter is air-dried and the DNA fixed or cross-linked on the filter, depending on the type of filter used.   

The filter is now ready to be processed for hybridization with a labelled DNA probe (cDNA or oligonucleotide) (radioactive or non-radioactive). When radioactive labelling with ³²P is used the autoradiogram is developed (e.g. Kodak XAR film) after appropriate exposure with intensifying screen at –80°C. It is easy to identify a positive clone and to carry out further analysis starting with the corresponding frozen clones as the individual colonies are well aligned and discrete.

The protocol for DNA screening is used for selection of modified plasmids but other systems can be applied as well.

At the end of the coding sequence of human b myosin heavy chain (bmhc) segment a specific immuno TAG (NH2-YYEEEYYEEE COOH) was introduced, against which there is a monoclonal antibody. In this way, a specific detection of the corresponding protein product after transfection in muscle cells is accomplished. After insertion of a 34 bp segment, encoding the TAG at the 3’-end of the bmhc coding sequence in an expression plasmid, colonies were screened for correct insertion of the radiolabelled TAG oligonucleotides.

Results for one MicroWell plate 96F with two positive controls in the bottom (Fig 2).

Fig. 2
DNA screening for selection of modified plasmids.
Positive clones are A1,10; B4,6,7; C6; E6, 7; F4, 10; G5, 10; H8, 9.At the bottom two positive controls are shown.

A. Dot Blot of bacterial clones
Procedure is the same as for in situ DNA screening previously presented, except in this case an inducible expression vector is used (e.g. a temperature inducible expression vector such as the pEX vectors).

B. Induction of recombinant protein expression on filters
Induction of expression must be made on the filter after step I.A.4 by adding the specific inductor (IPTG for example) or by switching temperature from 30°C to 42°C if you use a pEX vector, dependent on the promoter used for expression.

C. Bacterial Lysis on filter
1. The lysis is performed by placing the filter containing bacterial colonies face up (Nitrocellulose Millipore HATF) on three sheets of Whatman 3MM paper previously soaked in 5% SDS followed by heating in a microwave oven for one minute at full power (approximately 650 W).   

2. SDS is then removed by an electrotransfer in Tris 20 mM, Glycine 160 mM, in a 2x2 Whatman sheet sandwich, at 50 volts for 30 min. Place the colony side facing the negative electrode.   

3. Blockage of non-specific binding is performed by overnight incubation at 4°C in TBS-Tween containing 1% BSA and DNase I grade II at 10 mg/ml. The filter can then be treated in an ordinary Western blot procedure (e.g. primary antibody incubation, washings, secondary antibody coupled to Phos.Alk. or peroxydase incubation and revelation with the adapted substrates for colour development).