help on lambda maxi preps

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue May 23 10:44:54 EST 1995


Richard (rlconverse at delphi.com) wrote:
 
> 1. I never have any trouble setting up a liquid lysis culture using the
following method: pick one plaque from a freh plate into 10 mL of NZY in a 50
mL screwcap Fisher conical centrifuge tube, shake overnight @ 37 degrees, spin
down & purify the lysate th
e next morning. When I attempt to "scale up this procedure by picking more
plaques into a larger volume of liquid culture, it invariably fails (I see no
lysis, only bacterial growth--and these cells fail to lyse upon addition of
chloroform!).
 
> 2. I have attempted to set up liquid lysis cultures using a variety of
protocolsand have never seen lysis on a maxi scale. Does anyone out there have
a good method for isolation of DNA from lambda phage maxi-preps that
consistently works?               Richard--

And Vince Schoenfeld replied:

>>I think you're underestimating the importance of phage/cells ratio. I found
>>it was somehow more of a problem with maxi-cultures (the old toothpick 
>>technique
>>seems to be fine with minis).
>>Try and make sure you have about 10 to the eight pfu for a 500 ml 
>>prep at an OD
>>which corresponds to approx twice the pfu. 
>>You'd have to determine what OD corresponds
>>to what cell concentration. I know it all 
>>sounds a hastle but it only takes a day to determine
>>the titre of plate lysate. 
                             
Yes, this is a notorious problem with lytic lambda growths.  The problem
stems from the fact that the phage numbers are expanding faster than the
bacterial numbers, but the bacteria become refractory to infection when
they pass out of log phase.  So if you put in too little phage, the bacteria
become refractory before the phage overtake them and lysis is never observed.
If you put in too much phage, they overtake the bacteria before there is
enough turbidity to really see, so you don't notice the lysis.  Some 
fraction of the bacterial are resistant to infection and so these continue
to grow and eventually you see the culture grow turbid, but there's no
appreciable phage titer or apparent lysis.  Even if you get the ratio
approximately right, it often happens that you clear about half the bacteria
just as the culture is going refractory.  At that point you have a high
phage titer, but without a practiced eye you may not notice the lysis.
What happens next is the phage expend themselves trying to infect the
refractory bacteria and your titer drops.  If you follow these growths
with a spectrophotometer, you can see the transient drop in OD and harvest
for a good yield; but if you just let it go overnight, you'll get a poor
yield in the morning.

To make it that much harder, your insert size influences the phage burst 
size which influences the rate the phage overtake the bacteria.  So there
is no magic ratio that works for all phage clones.  The only thing that
works uniformly for me is to start 4 cultures with moi spanning about
a 10 x range around the supposed optimum ratio and then one of them
usually lyses on schedule (about 6 hr) and gives a good yield.  We usually take
4 x 10^8 pfu and 4 x 10^10 cells as our starting point for a 1 liter
culture (assuming 1 OD600 = 8 x 10^8 cells).  We also mix the phage
and the cells in a small volume with Mg and incubate at 37 to get
the first round of infection going.  Note that the phage/bacteria
ratio is not constant throughout the growth; so if you decide to start
with a higher bacteria concentration to move things along, then you
need an increased ratio of phage.  I'd guess about 10x more phage for
each doubling of bacteria.  

The biggest puzzle to me in all this is why the mini preps don't display
all these complexities; but it's a long standing observation that mini
preps are forgiving of initial conditions, and maxi preps are not.

Someone mentioned plate lysate preps, which is an excellent alternative to
the above.  If your moi are way off, then it's sort of obvious from the
appearance of the plates.  Also, the phage don't diffuse around much, so
once produced they don't go away.  The only drawbacks are kind of minor:
1) It's logistically hard to go to very large scale.
2) You have to be more fastidious about cleaning up the prep., or else
    you'll get crud from the agar into your DNA an it'll inhibit your
    enzymes.

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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