2xYT Agar
LAB 180
Description
An agar version of 2xYT broth, for the growth of host cells of
filamentous single stranded bacteriophages e.g. the M13 phage.
Formula
g/litre
Tryptone
16.0
Yeast extract
10.0
Sodium chloride
5.0
Agar
15.0
Method for reconstitution
Weigh 46.0 grams of powder and disperse in 1 litre of deionised
water. Allow the mixture to soak for 10 minutes, swirl to mix and
sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C, pour
into sterile Petri dishes and allow the medium to set. Dry the surface
prior to inoculation.
Appearance: Straw, clear gel.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Escherichia coli DH5
(ATCC
®
53868).
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in
the dark.
Inoculation: dependent upon application.
Incubation: 37°C aerobically.
2xYT Broth
LAB 179
Description
A nutritious liquid medium formulated to promote the growth of host
cells, thereby encouraging increased replication and yield from
filamentous single stranded bacteriophages (such as the M13 phage).
Formula
g/litre
Tryptone
16.0
Yeast extract
10.0
Sodium chloride
5.0
Method for reconstitution
Weigh 31.0 grams of powder and disperse in 1 litre of deionised
water. Allow the mixture to soak for 10 minutes, swirl to mix and
sterilise by autoclaving at 121°C for 15 minutes.
Appearance: Straw, clear liquid.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Escherichia coli DH5
(ATCC
®
53868).
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20°C in the dark.
Inoculation: Mix fresh overnight culture of host cells with
bacteriophage and use to inoculate 2xYTBroth.
Incubation: 37°C aerobically, typically for 4-5 hours to reduce risk
of selecting deletion mutants.
Interpretation: Concentrate bacterial cells by centrifugation and
transfer supernatant containing bacteriophage to a fresh tube, being
careful not to disturb the pellet formed. The resulting bacteriophage
stock can be stored at +4°C or -20°C.
3.6
NE W
NE W
