Library should be amplified right after the packing, which greatly increases the number of copies of the library. However, the compositions of the library may have potential changes due to the different growth rate in cloning. This change in composition ratio can be minimized by pre-adsorbing the library onto bacteria and using high-density plate and short-term culture.
Method and Material
Reagents & Kits Agarose LB NaOH SSC Tris•CI
Materials Nitrocellulose membrane filters Vacuum oven
1. Determine the titer of the phage libraries with serial dilution.
Note: Different vectors should use proper bacterial strain (e.g., P2 lysogen for EMBL vectors). LB plates should be prepared and dried several days before plating.
2. Mix the bacteria host of plates and recombinant phage in a culture test tube (Table 1) and incubate at 37 ℃ for 20 min.
|LB plate ingredient||Size|
|82mm||150mm||245mm x 245mm|
|Phage (pfu)||5 000||20 000 ~ 30 000||150 000|
|Top agarose (ml)||3||7||30|
3. The number of plates in library screening is decided by phage number on each plate.
4. The number of phages on each plate depends on the number of recombinant phages in the library and the appearance probability of targeted clones.
5. Add 0.7% top agarose into the test tube, quickly pour them onto the heat retaining plate. Culture it at 37 ℃ for 6~12 h till the plate is covered with plaques. These plaques should be in proper size and not connect with each other.
Note: The top agarose should be cooled to 45° to 50°C before use. The bacteria will be killed if the top agarose is too hot, while the library will solidify in the tube if it is too cold.
It is crucial to incubate the phage plaques for a correct amount of time. There are two reasons. Firstly, the phage plaques should be large enough so that they are able to contain adequate DNA to show a good signal. Secondly, it is difficult to purify if the phage plaques are too large and become confluent. Because most of the nucleic acid probes give quite strong signals, we prefer weaker ones and smaller plaques.
6. Place the plate at 4 ℃ for more than 1 h before it is used in the filter membrane photocopying experiment.
7. Mark on the nitrocellulose membrane with a ballpoint pen and make it face up.
Note: Use forceps or gloved hands only to handle nitrocellulose filters.
8. Place the filter membrane on the chilled LB plate covered with plaques for 1 ~ 10 min and mark the positions by puncturing with 20-G needles.
Note: To better mark the filter and agar, some researchers dip the needle in India ink. Other researchers mark on the back of the agar plate with black markers. Make two replicas from each filter, hybridize both to the DNA probe and compare the autoradiographs of the two replica filters to eliminate possible artifacts. It is very useful, especially for screening with an oligonucleotide probe.
9. Take out the filter membrane with a flat head tweezers, lightly face up on a piece of filter paper and dry it in room temperature for 10 min.
10. Face up the transferring side and place it one after another on three pieces of 3 MM filter paper soaked by the following three liquid for 1~2 min each.
(1) 0.2 mol/l NaOH / 1.5mol/l NaCl
(2) 0.4 mol/l Tris•Cl pH7.6 / 2×SSC
Note: The 3MM paper must be wet enough so that the filters can immediately reach saturation while not so wet to have the solution pooled on the surface.
11. Dry the filter membrane in a vacuum oven at 80 ℃ for 90~120 min, or place it in a 42 ℃ incubator overnight. If you don’t need to do hybridization experiments temporarily, you may clip the filter membrane with dry filter paper and keep it in room temperature.