Question: lambda plaques not transparent, bacterial lawn not , opaque

Michael Thompson mthom0 at pop.uky.edu
Thu Jan 25 17:12:29 EST 1996


>> My plates are a week old (instead of two days old)

>Yes, I have found that the age of the plate can affect the size of the
>plaques, because an old plate is dryer, and the top agar should be >enough
>wet to be really soft. But... it is not your problem.

					It is true that an older plate will yield slightly smaller plaques, but when screening (on a primary screen) it's important that the plaques aren't allowed to grow too large.  If they are too large you will end up performing an extra round or two of screening trying to purify your positive plaque.
					I've also found that older plates are better for screening, especially when using the 150mm plates.  The top agarose (or top agar) sticks to a drier plate much better and eliminates that pesky problem of your top agarose coming up with your filter when you try to lift it.  I'd much rather deal with smaller plaques than with sticky top agarose




>> I pour my top agarose at 52C instead of 48C (otherwise the agarose hardens too
>> quickly)

>Not very important. When you use very hot top agarm what happens is that
>most of the cells die, and you get.... isolated colonies instead of a lawn.

						Again, this one depends on whom you talk to.  I do mine at 49C.  Another student in the same lab uses 47C, and yet another person I have talked to does hers at 48C.  But it shouldn't be a problem, especially if you pour the bacteria/phage/agarose mixture quickly enough.

>> I use ~10 ml of top agarose per 150 mm plate (they recommend 8 ml)

>Idem. I always use more because can be spreaded more easily.

I always use 8 mL per 150mm plate, because if I use any more top agarose the plaques can get slightly "buried" in the top agar and you won't get a good filter lift.


     Another suggestion:  The protocol that I use directs that the host strain be grown in LB supplemented with 0.2% Maltose and 10 mM MgSO4.  I also add magnesium to the top agarose, and to the cells once I have spun them down and resuspended them at the correct density (I've found that I get very repeatable results if I resuspend the host cells at 0.5 OD in 10 mM MgSO4)

Hope this helps.

Michael Thompson
University of Kentucky
Department of Biochemistry




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