I have no much experience on that, but even though I know this is the classical procedure you culd try to propagate helper phage by infecting exponentially growing TG1 and growing them on kanamycin plates. You will obtain colonies (not plaques) that can be propagated in liquid media, giving rise to helper phage that you can purify. This aleternative procedure usually gives good results.

Make sure the strain has not lost the F' episome by streaking it first on minimal medium plates. It does not happen frequently, but it does happen.

Also, do you have any idea of the titer of the infecting phage? I have never worked with KM13 but the titer of 'normal' M13 stocks can be quite high, so you have to dilute them appropriately before you can observe individual plaques.

Hope it helps!

Thank you so much Gertrudis Rojas, I got your point, that only those bacteria will give colonies that will contain KM13 phage inside it because it has Kanamycin resistance gene. but my point is that, how can the bacteria infected with phage give colonies, because KM13 infection leads to lysis of bacteria. Please correct me if I am wrong.

Thank You Alejandro Martin

Yes, I have first streaked the TG1 on M9 minimal medium then made stock from it by inoculating single colony into 2XTY medium. from the stock I did primary and secondary culture, so I hope that there will be no loss of F' episome.

and , I have not checked the PFU, actually I have taken the KM13 phage from one of my senior and she said that she stored this phage at -80 degree celsus around 1 year 6 months back. So, is it possible that the phage might have lost his infection efficiency ?

Sanjay Kumar , filamentous phage do not lyse the host bacterium. When you plate M13, f1 or related phages what you get are not plaques in the classical sense of the word, but zones of diminished growth that may be hard to see. Hence Gertrudis Rojas 's suggestion of plating the infected bacteria on selective medium to isolate phage-producing colonies.

What I was getting at, on the other hand, was that if the titer of the phage is, say, 10EXP11 pfu/mL (and this is not hard to get with filamentous phage), and you mix 1 uL of this -approx. 10EXP8 pfu- with 100 uL of an exponential E. coli culture -approx. 5*10EXP6 cfu- essentially all cells of the inoculum will be infected; hence you will not get individual plaques, but a continuous lawn of infected bacteria. You have to titrate the phage, that is, make serial dilutions and then use those dilutions to infect the target strain to find out the dilution at which individual plaques are clearly visible.

Hope it helps!

1- serail dilutions 2- you can also adjust the incubation temperature to 25 degrees fro two days. Also negative control cells with our phage are they growing or not? Also check the optical denisty. Thanks and Good luck. You can also add glucose 1% to the medium.

Yes, with M13 you dont see lysis plaques, but plaques of slower growth. If you add ifected bacteria on kanamyin plates you geshould t c olonies.

Thank You Gertrudis Rojas and Heba Nasser

Hi Gertrudis Rojas , As you suggested I have got colonies on kanamycin plates. can you please guide from here, how exactly I can propagate ?

1. I have to inoculate a single colony in 2XTY medium supplemented with kanamycin then incubate at 37 Degree celsius for O/N

2. then, this primary culture will be diluted in 500 ml 2XTY supplemented with kanamycin (Somewhere I read kanamycin have to be added after 2 hr of incubation)

please make me clear what exactly I have to do at this step and after this for how many hours I have to incubate?

I cultivate the colonies overnight at 28 degrees in 50 ml 2XYT plus 70 micrograms/mL kanamycin. Next day I inoculate this culture in a total volume of 300 mL fresh 2XYT plus kanamycin and grow it overnight to have more helper phage. You can vary the scale and 37 degrees should also be ok, but thats what I normally do.

Thank you Gertrudis Rojas