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M13 phage amplification,who canhelp?


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#1 anonymous

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Posted 25 May 2001 - 09:00 PM

I want to amplify the M13 helper phage,after I incubate M13 and XL1-blue,and mixed them with the top agar containing X-gal and IPTG,I plated them on the LB plate.But the problem is ,after hours,even days,nothing heppens,no white ,no blue plaque.Nothing.The phage was provided from Japan,I don't know if M13 was damage in the ordinary mail posting period.What should I do? If M13 are all dead,can I extract the DNA instead?Thanks,I am eager to know the answer.

#2 melinda0711

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Posted 25 July 2002 - 07:51 PM

I replied here before but now it's gone. Anyway, i know this is an old post but someone else may wanna know.
I don't want to insult you by giving to simple of an answer, but did you add e.coli cells to the top agar too so the phage would have cells to infect and replicate in?

#3 danielxu

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Posted 31 July 2002 - 11:55 PM

I also experienced such circumstance before. In my opinion, the M13 helper phage wouldn't die coz it's quite tough since I also received even a phage library before via ordinary mail and it worked, there's no doubt.
My my perspective, the problem may be:
- the temperature of your top agar is too hot, i.e. far
 >>45 degree and the XL1-blue cell may die!!! (or maybe you've already prepare a stock of top agar and use simply by microwave the top agar then put it into the water and use within a short period of time! so the temperature would probably unfavable to the growth condition of the E.coli.

Maybe you can try using lower temperature top agar. i.e. 45 degree even it's quite readily solidified.





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