Large scale P1 prep

Dan Burgess Daniel.L.Burgess at um.cc.umich.edu
Thu Dec 8 11:44:44 EST 1994


Several people have been looking for an efficient method for prepping
large amounts of P1 clone DNA.  This protocol provides an average of 1
mg of high mol wt. DNA per 1 L culture: 

Large Scale P1 Prep:

1)	Innoculate a single P1 colony into 30 ml LB medium (25 ug/ml kanamycin)
  	and incubate overnight with shaking at 37¡C.

2)	Next morning, split the overnight culture into each of two 2 L flasks
  	containing 500 ml LB (25 ug/ml kanamycin).  Incubate with shaking at
  	37¡C for 1.5 hrs.

3)	Add IPTG to a final concentration of 0.5 mM.  Continue growth for an 
  	additional 5 to 7 hrs.  Longer (or overnight) growth lowers yield and
  	may induce deletions.

4)	Transfer 500 ml of culture to two 250 ml centrifuge bottles and chill 
  	on ice for 10 min (store remaining 500 ml at 4¡C temporarily).  
	
5)	Centrifuge the two bottles 20 min, 4K rpm, at 4¡C.  Pour off and
   discard supernatant.

6)	To each bottle (with pellet) add another 250 ml of remaining culture 
	  (was at 4¡C) and centrifuge again 20 min, 4K rpm, at 4¡C.  Pour off 
	  as much supernatant as possible.

7)	Resuspend* each pellet in 5 ml of ice cold: 
 	50 mM glucose
		10 mM EDTA
		25 mM Tris-HCl (pH8.0)

	*(Use a wide-bore pipette if necessary and resuspend gently to prevent
	  contamination of P1 with E.coli genomic DNA)

8)	Transfer each suspension to a seperate Oakridge tube and add 1 ml of 
  	fresh lysozyme solution (12 mg/ml).  Mix gently.  Incubate in ice-water
  	bath for 20 min.

9)	To each, add 12 ml of freshly prepared:
  	0.2 M NaOH
	 	1% SDS
	
	Mix thoroughly by inversion and incubate in ice-water bath for 10 min.

10)	Add 7.5 ml of cold 3 M Na-acetate (pH 4.6).  Mix carefully by
inversion.
	   Incubate in ice-water bath for 20 min.

11)	Centrifuge 30 min, 15K rpm, at 20¡C (room temp is OK).  

12)	Transfer supernatant to a 50 ml screw-cap tube (Corning, orange-cap). 
 	  Sometimes residual SDS can be seen to precipitate at this stage.
	   Ignore it.  

13)	Add 100 ul RNase (10 mg/ml) and incubate at 37¡C for 20 min.    

14)	Extract 2 times with 12 ml phenol-chloroform (or until clean
interface),
	   then extract once with 12 ml chloroform-isoamyl alcohol (24:1).  Gentle
 	  vortexing to mix during the extractions is OK at this point because the

   	P1 should be mostly supercoiled and any E.coli genomic contamination
    that	will occur, already has.  Centrifuge after each extraction 10 min
    @ 3K at room temp.  

15)	After the last extraction, split each supernatant into two Oakridge 
    tubes and add 2 volumes of cold 100% EtOH to ppt.  Leave overnight 
    at -20¡C.  (There should be a total of 4 Oakridge tubes at this
    point).

16)	Centrifuge 30 min, 15K rpm, at 4¡C.  Pour off supernatant (save until
    you check final yield).  Use a marker to make a dot on the tube where
    the pellet is.

17) Put 2 layers of parafilm over the tops of Oakridge tubes with the
    pellet	inside.  Poke a few small holes in parafilm.  Dry pellets in
    a vacuum for	@ 10 min.

18)	Resuspend the pellets in TE or Water to a total volume of 1.5 or 2 ml.

	 I usually add about 0.5 ml to the first of the 4 Oakridge tubes and
 	pipet the pellet up and down until it's mostly dissolved.  I then 
	 transfer this solution to the second tube and repeat the process through
 	all four tubes.  Store this in an eppendorf.  It contains most of the 
 	DNA.  I then add a second 0.5 ml to the first tube, and repeat the
 	process through all 4 tubes to retreive any remaining DNA.  A third round
 	with 0.5 ml gives a final volume of 1.5 ml that contains virtually all 
 	the DNA.

Additional notes

* The DNA is usually yellowish or even dark yellow sometimes.

* There is often undissolved material after the final resuspension. 
  It doesn't seem to interfere in any subsequent reactions.  It's probably
  un-resuspended RNA and protein, which brings us to the next note...

* Even with the RNase step in this protocol, there is usually going to be
  a LOT of e.coli RNA remaining mixed in with the P1 DNA.  I wouldn't
suggest
  a longer RNase step within the protocol.  I just add a little to any
  subsequent restriction digests using the P1 DNA.  Alternatively, you can
  add an RNase step and set of extractions after the final resuspension. 
  A final RNase step and set of extractions yields DNA clean enough to
  give really good results on things like FISH (flourescence in-situ
  hybridization) of metaphase chromosome spreads.

* For determining the approximate yield, I avoid spectrophotometry
(probably
  because our spec is about 20 years old and doesn't like me).  I prefer to
  set up a a series of test-digests with a six-base restriction enzyme 
  (EcoRI or such).  For example: 1 ul, 5 ul, 20 ul P1 DNA by EcoRI and 
  RNase, and maybe a couple of digests without RNase to see just how much
  RNA actually is there.  Run these out on a 1% agarose gel.  It's also a 
  good idea to do this to verify the complexity of the P1 to make sure it
  hasn't deleted.  

* Pellets can be frozen overnight at -20¡C after step 6 if desired, and the
  protocol finished the next day.


Daniel.L.Burgess at um.cc.umich.edu 
U of M Dept. of Human Genetics, Ann Arbor
 
Positional cloning...it's not the kill, it's the thrill of the chase.



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