19 cDNA Library Screening
297
Preparation of ready to plate phage suspensions
Startingwith cDNA libraries or other phage suspensions ofknown titer (i.e.,
known nurober of phage particles per rol), dilutions in A.-dil are prepared to
achieve the desired nurober of phage per plate. For initial screening 5 x 10 3
phages per 100 111 areideal for each 90 roro plate.
Plating
1. The required nurober of LB plates are placed in a 42°C incubator for
prewarroing.
2. LB top-agar (roinirouro of 2.5 rol for each plate) is roelted in a roicrowave oven and then placed in a water bath at 49°C.
3. Foreach of the plates in the incubator one 12 rol screw cap tube is placed
in a suitable rack at rooro teroperature.
4. 100 111 of the host bacteria suspension in A.-dil is pipetted into each of the
screw cap tubes.
5. 100111 of diluted phage suspension is added to each tube and roixed with
the bacteria by brief vortexing.
6. Tubes containing bacteria and phages (infection roixes) are incubated
at 37°C for 25 roinutes, then placed at rooro teroperature.
7. Using a sterile 10 rol glass pipette which has been briefly prewarroed in
the flaroe of a Bunsen burner, 2.5 rol roelted top-agar is pipetted froro
the stock at 49°C to one of the infection roixes.
8 .. The contents of the tube are iroroediately roixed by rolling several tiroes
between the palros and are then poured evenly onto a prewarroed LB
plate.
9. The poured plate is left to stand upright on a flat surface at rooro teroperature until the top-agar has solidified (at least 5 roinutes).
10. Steps 7 to 9 are repeated for each of the infection roixes until all of the
roixtures of bacteria and phages are plated.
11. Plates are placed in an incubator at 42°C (for A.gtll) until plaques develop.
297
Preparation of ready to plate phage suspensions
Startingwith cDNA libraries or other phage suspensions ofknown titer (i.e.,
known nurober of phage particles per rol), dilutions in A.-dil are prepared to
achieve the desired nurober of phage per plate. For initial screening 5 x 10 3
phages per 100 111 areideal for each 90 roro plate.
Plating
1. The required nurober of LB plates are placed in a 42°C incubator for
prewarroing.
2. LB top-agar (roinirouro of 2.5 rol for each plate) is roelted in a roicrowave oven and then placed in a water bath at 49°C.
3. Foreach of the plates in the incubator one 12 rol screw cap tube is placed
in a suitable rack at rooro teroperature.
4. 100 111 of the host bacteria suspension in A.-dil is pipetted into each of the
screw cap tubes.
5. 100111 of diluted phage suspension is added to each tube and roixed with
the bacteria by brief vortexing.
6. Tubes containing bacteria and phages (infection roixes) are incubated
at 37°C for 25 roinutes, then placed at rooro teroperature.
7. Using a sterile 10 rol glass pipette which has been briefly prewarroed in
the flaroe of a Bunsen burner, 2.5 rol roelted top-agar is pipetted froro
the stock at 49°C to one of the infection roixes.
8 .. The contents of the tube are iroroediately roixed by rolling several tiroes
between the palros and are then poured evenly onto a prewarroed LB
plate.
9. The poured plate is left to stand upright on a flat surface at rooro teroperature until the top-agar has solidified (at least 5 roinutes).
10. Steps 7 to 9 are repeated for each of the infection roixes until all of the
roixtures of bacteria and phages are plated.
11. Plates are placed in an incubator at 42°C (for A.gtll) until plaques develop.