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JOHANN PETER HOSSLE
Subprotocol 1
Plating of a A.gtll cDNA Library
Materials
Media
• LB (Luria-Bertani) medium (per 1liter):
10 g Bacto-tryptone
5 g yeast extract
10 g NaCl
adjust pH with NaOH to 7.5; autoclave immediately
• LB plates: Add 7.5 g Bacto-agar (DIFCO) to 0.5liter LB medium and autoclave. Cool hot agar solution by placing in a water bath until the temperature is below 60°C. Alternatively store agar solutions on the shelf
at room temperature and remelt (melting temperature of agar: 47°C)
in the microwave oven before use. Pour melted agar into petri dishes
(0.5 liter agar solution for 20 to 25 90 mm dishes). Cool the plates in
a laminar flow work bench for at least 30 minutes. Let plates dry further
by leaving them at room temperature overnight. Dry plates are stored in
the cold room at 4°C until used.
• LB top-agar: 0.72 to 0.75% (w/v) Bacto-agar (DIFCO) in LB medium
• A.gt11 cDNA library: titrated in A.-dil
• A.- dil: 10 mM Tris-Cl, pH 7.5; 10 mM MgS04; autoclaved
Procedure
Preparation of host bacteria
1. 2 x 5 ml LB-medium are each inoculated with a single colony of E. coli
host strain (Y1088 for plaque-hybridization screening, Y1090 for immuno-screening) and incubated at 37°C overnight with constant agitation.
2. Centrifuge overnight cultures for 5 minutes at 3,000 x g
3. Resuspend cell pellets in 2 ml A. - dil each.
4. Cell suspensions can be stored at 4°C for up to one week.
JOHANN PETER HOSSLE
Subprotocol 1
Plating of a A.gtll cDNA Library
Materials
Media
• LB (Luria-Bertani) medium (per 1liter):
10 g Bacto-tryptone
5 g yeast extract
10 g NaCl
adjust pH with NaOH to 7.5; autoclave immediately
• LB plates: Add 7.5 g Bacto-agar (DIFCO) to 0.5liter LB medium and autoclave. Cool hot agar solution by placing in a water bath until the temperature is below 60°C. Alternatively store agar solutions on the shelf
at room temperature and remelt (melting temperature of agar: 47°C)
in the microwave oven before use. Pour melted agar into petri dishes
(0.5 liter agar solution for 20 to 25 90 mm dishes). Cool the plates in
a laminar flow work bench for at least 30 minutes. Let plates dry further
by leaving them at room temperature overnight. Dry plates are stored in
the cold room at 4°C until used.
• LB top-agar: 0.72 to 0.75% (w/v) Bacto-agar (DIFCO) in LB medium
• A.gt11 cDNA library: titrated in A.-dil
• A.- dil: 10 mM Tris-Cl, pH 7.5; 10 mM MgS04; autoclaved
Procedure
Preparation of host bacteria
1. 2 x 5 ml LB-medium are each inoculated with a single colony of E. coli
host strain (Y1088 for plaque-hybridization screening, Y1090 for immuno-screening) and incubated at 37°C overnight with constant agitation.
2. Centrifuge overnight cultures for 5 minutes at 3,000 x g
3. Resuspend cell pellets in 2 ml A. - dil each.
4. Cell suspensions can be stored at 4°C for up to one week.