Helper phage

Helper Phage

The following is based on the λZAP cDNA synthesis manual from Stratagene. See also the Stockinger Lab
protocol “M13K07 Helper Phage Production.”

Stratagene provides two different helper phages with their λZAP-cDNA synthesis kit:
(1) ExAssist interference-resistant helper phage
(2) VCSM13
ExAssist is used to excise, in vivo, the pBluescript phagemid from the λ Uni-ZAP XR vector. To do this
efficiently and without contaminating the excised phagemid with the ExAssist helper phage, this
methodology utilizes the Stratagene E. coli strain SOLR. This is because ExAssist helper phage contains an
amber mutation (UAG) that requires an amber suppressor tRNA in order to grow. When plated on the
SOLR nonsuppressing strain (Su-) strain, only the excised phagemid is permitted to grow. However to
propagate (amplify) the ExAssist helper phage requires using the E. coli strain XL1-Blue MRF′ because
this strain harbors the supE44 mutation, which provides a glutamine suppressor tRNA. The VCSM13
helper phage is used strictly to produce single stranded DNA from the already excised phagemid. (This is
also what M13K07 is used for.)
Storing the Helper Phage:

Stratagene supplies both helper phages in 7% DMSO and recommends storing them at -80oC. As long as
there is a –80oC stock in the lab, the amplified lab prep can be stored at +4oC.
Titering the Helper Phage:

Streak out E. coli strain XL1-Blue MRF′ onto an LBTet15 plate.
Pick a single colony of XL1-Blue MRF′, inoculate into LB and grow to OD600 = 1.0. (No need to include the antibiotics in the overnight cultures.) Meanwhile prepare a serial dilution of the phage in TE buffer. Expected number of phage should be about 1010 pfu/ml; therefore dilute phage to create dilutions 10-4 to 10-7. 10µl (10-2) 100µl (10-4) 100µl (10-5) 100µl (10-6)

When preparing serial dilutions of bacteriophage, it is usually much more accurate to dilute 10µl into 100
to create a 10-1 dilution or 10µl into 1000µl to create a 10-2 dilution rather than to dilute 1 into 10µl or 1µl
into 100µl because one tends to obtain grossly exaggerated titers resulting from small pipetting errors. This
is particularly critical for the first dilution.
Add 200µl of XL1-Blue MRF′ cells at OD600 = 1.0 to individual sterile culture tubes (the number of phage
dilutions you plan to plate) in a test tube rack. Add 100µl of each serial dilution of helper phage to the culture tubes containing the XL1-Blue MRF′ cells. Place the test tube rack into a 37oC water bath for 15 minutes to allow the helper phage to attach to the cells. Meanwhile melt NZY Top Agarose in the microwave and allow it to cool to ~48/50oC. You need to pay close attention to the bottle containing the top agarose because it can quickly boil over; alternatively use a low power setting on the microwave. At the conclusion of the 15-minute incubation, add ~3 ml of the NZY top agarose to the test tube containing the helper phage & E.coli. Remove the tube from the rack (which is still sitting in the water bath) and give it a quick flick of your wrist mixing the contents and immediately pour it onto an NZY plate. (I usually do three plates at one by pipetting 10ml of the NZY top agarose from the bottle, dispensing ~3.3 to one tube, ~3.3 to second tube and then another ~3.3 to a third tube. I then pour the plates starting with the first tube I put the NZY into.) Allow the top agarose to cool (~5 minutes). Invert the plate and incubate overnight at 37oC. Count the number of plaques. Determine the titer; i.e., pfu/ml using the formula: (Number of plaques (pfu) X Dilution Factor) X 1000ul/ml
Where the volume plated (in µl) refers to the volume of the helper phage solution added to the cells.
Amplifying the Helper Phage:

Streak out E. coli strain XL1-Blue MRF′ onto an LBTet15 plate.
Pick a single colony of XL1-Blue MRF′ and inoculate into 10ml 2X YT and grow until OD600 = 0.3 (~2.5 x 108 cells/ml). (No need to include the antibiotics in the overnight cultures.)
Add helper phage at a multiplicity of infection (MOI) of 20:1 (phage-to-cells ratio). Do this for both
ExAssist and VCSM13.
(If amplifying VCSM13 helper phage, add kanamycin to a final concentration of 25µg/ml to the medium 30 minutes after the helper phage and cells have been allowed to grow together.) Grow the culture at 37oC with vigorous aeration (~300RPM) for 8 hours. Heat the culture to 65oC for 15 minutes. Spin down the cell debris and transfer the supernatant to a fresh tube. Titer the helper phage produced. ExAssist should be 7.5 x 1010 to 1.0 x 1012 pfu/ml and VCSM13 should be 1.0 x 1011 to 1.0 x 1012 pfu/ml Store at +4oC (Add DMSO to 7% for storage at –80oC).
Notes: Several steps greatly aid the ease and success of pouring good phage onto plates:
(1) Position the 37oC and 48oC water baths right next to each other with enough room in front of them for
you to work. Wipe this area down with 70% EtOH prior to plating.
(2) Use plates that have been prewarmed to 37oC (for one hour or more).
(3) Use sterile glass pipettes that have been prewarmed to ~50oC.
(4) Have a pipette holder in the work area.
Tet15 plates
Cool to 48/50oC Tetracycline (12.5mg/ml) 120µl
Tetracycline (12.5mg/ml)

(Tetracycline is light sensitive so keep plates in the dark)

NZY Agar Plates

NZY Top Agarose



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