smithson at nospam.opus.mco.edu
Mon Jun 1 16:25:48 EST 1998
Barbara Thompson wrote:
> I am trying to perform phage preps on isolated lambda gt11 phage. I have
> tried the plate lysate and culture methods in Maniatis as well as a slightly
> modified culture method. It seems that the cells I am using (LE392) take
> a while to grow, but once they do, never seem to lyse. Using the plate
> method, the DNA was degraded, and I could not use it for restriction
> digests. Does anybody have any suggestions or alternative protocols?
> barbt at bu.edu
It has been a while since I actually worked with lambda phages but during my
Ph.D. I ran into the same problems. It may be worth checking the E. coli
stocks by plating on minimal media or getting fresh ones from another lab. At
least twice low infectivity has been due to contamination of the stock with
other bacteria (usually bacilli).
Most of these methods were modified from Maniatis.
High Titer Phage Stocks
Essentially as described in Sambrook, Molecular cloning 2nd edition (2.65).
1) Plate 1/10th of a plaque suspension on 90 mm plate.
2) Incubate overnight at 37*C
3) Recover phage in 5 ml SM. Add 0.1 ml chloroform, cfuge 4000 x g, 10 min
4) Recover SN. Add 1 drop CHCL3. Store at 4*C. Yield 1010 pfu/ml.
Infection at high multiplicity
Again from Sambrook (2.72)
1) In 10 ml grow an overnight culture of the bacterial host.
2) In the morning incubate 500 ml of NZCYM with 1 ml of cells. ( I would set
this up at about 2 pm)
3) 37*C until A600 = 0.5 (this took about 3 hr., now it is 5 pm)
4) Add 1 ml of high titer phage stock
5) Incubate 37*C , 200 rpm until lysis occurs (another 3 - 5 hr., but often
6) Add chloroform (10 ml) incubate 10 mins)
7) Harvest culture proceed to purification.
Purification of lambda DNA
Similar to Sambrook (2.73)
1) Cool lysed culture. Add a pinch of powdered RNAse and DNAse (this means
you are adding more than 1µg/ml), enough powder to cover the tip of a small
spatula. (RNase - R4875 Sigma, DNase - DN25 Sigma, both of these are crude but
are sufficient for lambda preps).
2) Incubate at room temperature for 1 hour.
3) Add NaCl to 1M (14.6g/250ml), stand 1 hour on ice.
4) Centrifuge at 11,000 x g for 10 min.
5) Decant supernatant from debris, add solid PEG 8000 to s/n to 10%w/v dissolve
(this can sometimes take a while especially if you bought the cheap flakes of
PEG not the nice mol biol grade powder)
6) Stick in an ice bucket and leave in the cold room overnight.
7) Recover phage by cfuge 11000 x g , 10 min , 4*C.
8) Resuspend pellet in 8 ml SM per 500 ml original phage.
9) Extract 2 x with chloroform to remove PEG
10) Add EDTA to give 20 mM, pronase to 0.5 mg/ml or proteinase K 50 µg/ml
11) Add SDS to 0.5%, Incubate 1 hr at 37*C (pronase) or 56*C (proteinase K)
12) Cool to RT, Extract with equilibrated phenol (50 mM Tris pH 8.0) once,
phenol chloroform twice and chloroform isoamyl alcohol twice.
13) Precipitate the DNA with 0.1V NaAcetate 3M, 2V ethanol, at RT for 30 mins.
14) Centrifuge 12 000 x g 2 min 4*C. Redissolve in 500µl TE.
This used to give us almost a mg of phage DNA which was clean enough for
restriction digests without CsCl gradients. Using more RNase and DNAse in the
first step really cuts down on RNA contamination but you really have to add the
EDTA or your phage DNA will get chomped when the phage protein coat is digested
by the protease. If after two phenol chloroform washes you still have debris
at the interface then do it again and again until it is all clean, it really is
important to get rid of all the protein.
I am assuming that anyone reading this will be familiar with most standard
techniques in mol biol. If there is something missing that you don't
understand don't hesitate to contact me. Just remove nospam from the email
Hope this helps.
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