IG® Lambda Packaging Extract

Plating Bacteria Preparation:
The day before performing the packaging reactions, inoculate 50 mL of supplemented (10 mM MgSO4 ) LB broth with a single colony of the plating bacterial strain and shake overnight at 37 °C.
The day of the packaging reactions, inoculate 50 mL of supplemented (10 mM MgSO4 + 0.2% maltose) LB broth with 5 mL of the overnight culture and shake at 37 °C to an OD600 = 0.8-1.0. Store the cells at 4 °C until needed; cells may be stored for up to 72 hours.

Preparation of Lambda Packaging Phage:
Thaw the appropriate number of packaging extracts on ice. For every two packaging reactions, thaw one extract then place on ice. Note: Ensure that the extracts are completely thawed before use.
When thawed, immediately transfer half (50 µL) of each packaging extract to a second 1.5-mL tube and place on ice.
Add the substrate DNA (upto 10 µL [0.25 μg] of cloned fosmid DNA) to a tube containing 50 µL of extract. If performing an odd number of packaging reactions, the remaining 50 µL of extract can be refrozen at –80 °C.
Mix by pipetting several times; avoid the introduction of air bubbles. Return all of the contents to the bottom of the tube by brief centrifugation if necessary.
Incubate the reaction(s) at 30 °C for 90 minutes.
At the end of this incubation, add the additional 50 µL of thawed extract to each reaction tube at 30 °C (If performing only one packaging reaction, returned 100 µL of remaining extracts to –80 °C) and incubate the reaction(s) for an additional 90 minutes at 30 °C.
Add 1,000 µL of phage dilution buffer and mix by gentle vortexing.
Add 50 µL of chloroform and mix by gentle vortexing (store at 4 °C).
Assay the packaged phage by titering on the appropriate bacterial strain (for example IG® fosmid replicator strain).

Media and Solutions:
LB Broth (1 Liter): 10 g Bacto-tryptone, 5 g Bacto-yeast extract, 10 g NaCl, Adjust pH to 7.0 with NaOH
LB Plates: LB Broth with 1.5% (w/v) Bacto-agar
LB Top Agar: LB Broth with 0.7% (w/v) Bacto-agar
Titering Phage Extracts:
Make serial dilutions of the packaged phage in phage dilution buffer. Use 102, 104 and 106 dilutions for the control reactions.
102 dilution is 10 µL of packaged phage particles into 990 µL of phage dilution buffer; vortex mix.
104 dilution is 10 µL of 102 dilution into 990 µL phage dilution buffer; vortex mix.
105 dilution is 100 µL of 104 dilution into 900 µL phage dilution buffer; vortex mix.
106 dilution is 10 µL of 104 dilution into 990 µL phage dilution buffer; vortex mix.
Add 100 µL of the appropriate serial dilutions to 100 µL of prepared plating bacteria and incubate for 15 minutes at 37 °C.
Add 3.0 mL of melted LB top agar, supplemented with 10 mM MgSO4 and cooled to ~48 °C. Vortex gently and pour onto pre-warmed (37 °C) LB plates. Allow the top agar to solidify and then incubate inverted overnight at 37 °C.
4. Count the plaques and determine the titer (pfu/mL) and packaging efficiency (See sample calculations).

Titer calculations:
[(# of plaques) (dilution factor) (1000 µL/mL)]/[(volume of diluted-phage plated [µL]) (amount of DNA packaged)]
If 100 plaques are observed on the plate containing the 10⁶ dilution, the packaging efficiency is calculated as follows: (100 pfu) (106) (1000 µL/mL) / (100 µL) (0.25μg) = 4 × 109 pfu/μg of DNA.
Therefore, the packaging efficiency of this reaction is 4 × 10⁹ pfu/µg DNA.

References:
Hohn, E.G. (1979) Methods Enzymol. 68, 299.
Gunther, E.G. et al., (1993) Nucl. Acids Res. 21, 3903.
Kohler, S.W. et al., (1990) Nucl. Acids Res. 18, 3007.