Lysate prep: donor strain is inoculated overnight in LB then diluted 1:100 into 5 mL LB + 0.2% glucose + 5 mM CaCl2, incubated for 1 hour. 100 uL of P1vir was added, then incubated until total lysis was seen. 200 uL of chloroform was added, vortexed, and spun down. Supernatant was kept and lysate was titered (~5E8 PFU/mL for most lysates).
Transduction: 5 mL overnight is inoculated in LB. Stationary culture is pelleted and resuspended in 2.5 mL of 10 mM MgCl2, 5 mM CaCl2. 100 uL cells was mixed with between 1-100 uL of P1 lysate. Phage was allowed to adsorb for 30 minutes at 37 C, after which 1 mL of LB + 200 uL of 1 M NaCitrate was added and incubated at 37 C with shaking for 1 hour. Cells were pelleted then plated on selective plates containing 5 mM NaCitrate as well as spotted onto nonselective plates containing 5 mM NaCitrate (controls). I'm also using a control with no phage added.
All solutions are autoclaved.
I originally got a few phage transductions to work (confirmed via PCR) however newer attempts have not succeeded. No colonies on any of my selective plates. On my nonselective LB plate, I notice a full growth in the no-phage-control spot, but only a couple of colonies in my added phage spots-- it seems that there is a lot of lysis going on. There does not seem to be a dose dependent response depending on how much phage I add (ie 1 uL of phage lysate added shows same result as 100 uL phage lysate added). I don't think I have phage contamination because I use same solutions and steps for the no-phage-control.
I've tried re-making solutions a few times with no avail either. Most confusingly, I retried using the lysate preps that were successful for me in the past and was unsuccessful this time around. There was also too much lysis as seen by spotting onto nonselective plates.
I'm thinking the only variables that there could be are the solutions, the phage lysate, the handling, and the recipient strain. It seems to me that there should not be variation in the phage lysates as I had tried lysates that were successful for me, and recipient strains are also the same. So that leaves handling/execution and solutions. I have remade solutions a few times now, and I have not changed the way I do transductions so I'm at a loss.
What is my next step in troubleshooting? Does anyone have insights into what could be going wrong?
Your protocol seems fine. Could you add the genotype of your donor and recipient? and what strain background it is.
My experience is that nearly always the problem is the lysate. Sometimes adding too much will cause too much killing, so usually you try the lysate direct and 10 and 100 fold dilutions. But most often the problem is the lysate is too low titer. 5e8/ml seems pretty low. I would suggest trying new lysates.
your strain background will not cause any problems. When I had this problem before I did the following:
1- take 3-5 lysates that worked previously and mix in a separate eppie all of them in equal volume then use that for preparing a new lysate.
2- Grow the lysate on a wild-type K12 strain, MG1655 should work.
3- Use the lysate from the MG1655 to make a new lysate from the keio strain
4- Repeat the transduction.
In case your out of luck with the above, you might want to consider getting a new P1 stock either from a neighboring lab or a culture collection.
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