Did you try to titer your stock and determine the pfu before and after amplification? Usually you would expect to see some lysis but not always, depending on the phage and the strain. Also be sure the strain you are using is an appropriate host for that particular phage vector. 

Hi,

My recollection is that the MOI is important here. When you say "add phages" do you resuspend a plaque or control the number of phages added? MOI should be around 0.1 if I'm correct. I also seem to remember that adding maltose (to induce expression of the  lambda receptor) was not necessarily a good idea because you adsorb so many phages that it is no good for further phage replication. In any case Michael's suggestion have to be considered first!

In addition to the previous answers you may grow your control agar plates just longer to see if any plaques are forming at all.

yes we titred the amplified library. our aim is to use PCR screening to narrow down on a phage sub-pool rather than conventional filter methodology. the strain is correct since we purchased it. 

we add phages directly...no plaques

We used maltose as the protocol says so...isnt it needed for phage adsrption..how will it reduce phage replication?

yesterday we grew cells to an od of 0.3 (medium changed to NZY) with maltose and Mg++. Cells were resuspended in 10 mM MgSO4, and phages (SM buffer), added. after overnight growth we got lysis...turbidity of cells appeared 'cleared' and 'swirly material' was seen at the bottom. 

SO do we need to get to a cleared extract stage before we proceed to PCR...bcos only technically then the phages are released out...is that correct?...4-6 hours is not correct as per the protocol, atleast for us. 

So your principle of growing lambda phages on bacteria seems to work due to some clearing/lysis. Since one can perform PCR from many crude materials without the need to have clear starting material, I would expect that you can use any not so clear sample of your amplified library, perhaps you remove the bacterial debris with accurate filter to get a clear solution with lambda phages. The PCR reaction may be sensitive to different amounts of Mg++ ions, but that should be checked and adjusted to a usefull PCR buffer.

how do I reduce Mg2+? adding EDTA..? that will inhibit PCR also...

I just wanted to point to a possible difference in the recommended Mg++ concentrations, since I do not memorize it - maybe they are in a useful range and there is no need to adjust it. One way to reduce too high concentrations should be dialysis bags.

Are you certain that your cells are growing exponentially?  Did you do a growth curve?  An OD of 0.2 (NOT 2.0) , for E. coli, is in the early stage of exponential growth.  Make sure all of your reagents are at 37C, before adding them to the cells.  The titer seems to be the most likely culprit to me.  Perhaps practice with some phage that are not associated with the library until you get better results (i.e. clearing and plaques ).

A safe way to amplify a genomic lambda phage library is to plate and grow them on several agar plates (I think 8-24 was a good number of big 15cm plates), see the colonies/plaques and collect them in liquid supernatant... Long time I did this in the mid-1980s when the general methods book was just the "Maniatis" (originally one band, later three).

It seems you may have solved your problem buy now! But just to clarify my comment about maltose... Expression of the lamdba receptor protein LamB is induced in the presence of maltose. However basal expression in the absence of maltose is sufficient to provide a low number of receptor protein per cell, enough to accommodate adsorption with low MOI. If you add maltose and infect at higher MOI, many phages can infect a single cell and this is no good for phage development (I should have used "development" instead of "replication"!). I confess I haven't gone back to check the original publications about this, it's just what I remember from growing lots of lambda lysates through the years...

Hi Gayatri - It's all very empirical: if you need to incubate your culture overnight to get a full lysis you should stick with that. Depending on MOI, sometimes lambda overgrows E. coli (that's what you want), and sometimes E. coli overgrows lambda (not good!). I personally found that adding chloroform (1 ml/100 ml culture) and NaCl to 1M and incubating the culture at 42C for the last couple of hours helps lysis a lot. Good reference is Molecular Cloning: A laboratory Manual by Maniatis, Fritsch, and Sambrook - either 1st or 2nd edition is fine, and lambda protocols are in the very beginning of the book.

So Ariane, if I understand correctly, low MOI ensures,...you do not lose specific phages from a pool...ie you have a better chance of amplifying 'low' copy sequences or sequences otherwise not adequately represented in the library??

Thanks for all your comments...any ideas on how to proceed to PCR...since the lysed phages are in MgSO4 and that will affect PCR conditions...?

Continuing with the phage question, we managed to screen the library using PCR in the 96 well plate. We now have a new problem.

1.      In the primary Screen  we grew E. coli  to on OD-O.3.

2.      500 microlitre of this  e. coli with 500 microlitre of SM buffer + 10 microlitre of amplified phage library was incubated at grown at 370c for 15'.

3.      This 1 ml of e. coli with adsorbed phage was mixed with 9 ml of LB containing 10 mM MgSO4.

4.      200 microlitre of this was plated to confirm plaque formation  the rest was split into a 96 well format (8 columnsx 8 rows; 100 microlitre each well) for PCR screening and grown overnight (lysis was observed, slimy stringy). The plate was spun down at low speed and the lysate sed for PCR screening.

5.       Upon identifying   the well which gave the desired PCR product, the  plate was spun down and 10 microlitre of the lysate was used to infect e. coli grown as in (1.). Upon plating, following steps 2-4 listed above, only e. coli are seen on the plate, no phages….Why is this?

6.       The protocol for PCR screening of Phages (Israel) mentions that the plates with lysate can be stored for a month at  four degrees C

7.       Do we need to add chloroform to each well as in the phage amplification steps in standard protocol?….I do not think it is some other bacterium, as we have consistently observed this only in the secondary screening step when we plate the phages.

8.       Will spinning down the lysate drawn put from the PCR positive well, after addition of a drop of chloroform help?

not sure, what went wrong. The chloroform might be necessary to stop not only overgrowth by bacteria and fungi, but may also stop some enzymatic degradation. Since you lost your lambda phage after removing an aliquot for the PCR process, I suggest you just remove a second aliquot (or split the one for the PCR and store it) - and use this later for plating on agar to directly see the colonies. After identifying the clone you may just grow it as plaques on agar plates which you then could store long enough to solve other apsects. One can extract lambda phages easily from agar plates.

Hi Robert

Thanks. the only difference between what we use for plating and what we use for PCR is the time of incubation. the phage were plated  15 min after incubation with e. coli while the lystae for PCR was obtained after overnight growth. will try what you said however.