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All your clones should have been archived as a DMSO stock. Your regrididng records should allow you to correleate the sequenced clone to the original DMSO archive.The phage can be rescued from the DMSO archive by re-infection of XL1-Blue cells in 2 different ways individual plaques or streak
Rescue protocol - individual plaques
(1) Prepare XL1-Blue cells in 10mM MgSO4, NZY top agarose at 50oC and prewarmed NZY plates as described previously (protocol)
(2) Use a sterile hypodermic needle or gilson pippete tip to take a small scrape from the frozen DMSO. Do not allow the DMSO stock to defrost (store it on dry ice while it is out of the -80oC).
(3) Add the scrape of DMSO stock to 200ul XL1-Blue cell from step1
(4) Incubate cells at 37oC for 20 min to allow phage to attach.
(5) Add 3mls NZY top agarose 50oC to the infected cells, mix and plate out onto the prewarmed NZY plates. Gently swirl the NZY top agarose around the plates before it sets to ensure even distribution of plaques (nb. NZY top agarose sets at 42°C). Allow NZY top agarose to set .
(6) Invert plates and incubate overnight at 37oC
Day2
If you do not obtain well separated plaques you may need to repeat the above procedure infecting the cells with less DMSO archive. If well separarated plaques are obtained pick several plaques and prepare a PSB stock. The plaques should be picked a soon as possible to prevent the possibility of cross contamination due to phage diffusion.
1. Using sterile yellow tips core the centre of 2-3 plaques. A small volume of agarose/fluid should picked up by the tip. Transfer the tip to a sterile eppendorff containing 25 ul PSB.
2. Allow the phage to diffuse into the PSB for approx 20 min at room temp.
3. Remove the tip and mix well to ensure dispersal of phage. This is your fresh PSB stock
This PSB/phage stock can be stored at 4oC for several months if chloroform is added PSB:Chloroform (50:1). This stock can be used as a viable source of phage. If the phage/PSB stock is frozen at anytime the phage will be unlikely to be viable on thawing and will only be useful as a template for PCR.
You should expect all plaques to be clonal however it is important to check the identity of any rescued phage before processing it further.
From this PSB stock you can:
PCR the insert and sequence from the PCR product
Excise the pbluesript plasmid from the phage and sequence from the plasmid. nb.all the LambdaZapII lamda UniZap clones can be excised ands maintained as plasmids in SOLR cells (see excision protocol)
Resue protocol -streak on lawn of XL1-Blue cells
This method allows you to prepare larger volume stocks and rescue several phage on a single plate .
(1) Prepare XL1-Blue cells in 10mM MgSO4, NZY top agarose at 50oC and prewarmed NZY plates as described previously (protocol)
(2) Add 3ml of NZY top agarose 50oC to 200ul of XL1-Blue cells. Mix gently and plate out onto a pre-warmed NZY plate. Gently swirl the NZY top agarose around the plates before it sets . Allow top agarose to set.
(3) Using a sterile hypodermic needle or gilson pippete tip to take small scrape from the frozen DMSO. Do not allow the DMSO stock to defrost (store it on dry ice while it is out of the -80oC). Touch the needle to the top agarose or gently make a small streak (1cm across the plate). Allow the DMSO scrape to diffuse into the top agarose.
(4) Invert plates and incubate overnight at 37oC
Day2
You should see a large zone of cell lysis around the area where you placed the DMSO stock streak. Prepare a PSB stock.
(1) Scrape the area of top agarose containig the zone of cell from the plate into 300ul PSB.
(2) Vortex briefly and allow the phage to diffuse out of the agarose at room tmeperature for 30min.
(3) Pellet agarose by centrifugation at 13k 2min. Transfer the phage/PSB supernantant to a fresh tube.
This PSB/phage stock can be stored at 4oC for several months if chloroform is added PSB:Chloroform (50:1). This stock can be used as a viable source of phage. If the phage/PSB stock is frozen at anytime the phage will be unlikely to be viable on thawing and will only be useful as a template for PCR.
This PSB/phage stock will not be clonal and may contain more than one phage if your stock was contaminated in any way. You should check the stock by PCR.
From this PSB stock you can:
PCR the insert and sequence from the PCR product
Excise the pbluesript plasmid from the phage and sequence from the plasmid. nb.all the LambdaZapII lamda UniZap clones can be excised and maintained as plasmids in SOLR cells (see excision protocol)