help problem with lambda

suter at VAX.mpiz-koeln.mpg.d400.de suter at VAX.mpiz-koeln.mpg.d400.de
Tue Jan 11 09:19:20 EST 1994


here is a method to isolate dna from lambda:

1.7		Lambda quick prep from lysed plates
Clemens Suter-Crazzolara, 1993

Materials
DNAse I		(Sigma DN-25, 10 mg/ml,store in 50 % glycerol at -20 oC)
RNAse A		10 mg / ml (Sigma)
10 x pk buffer 	0.1 M Tris 7.8; 0.05 M EDTA; 5 % SDS
It is essential that the LB plates do not contain agar, but agarose of
good quality (like FMC): Agar contains inhibitors for enzymes which are
difficult to get rid off.

Method
1.	grow lambda phages confluent on a 11 x 11 square LB/agarose plate.
2.	scrape of top agarose, put in tube.
3.	add 8 ml 10 mM MgSO4 and 40 ul chloroform, shake gently 5 minutes
4.	spin 30' 4 k.
5.	add to supernatant 0.2 ug / ml DNAse1 and 1 ug /ml RNAse A
6.	spin solution 30 k 30' in SW 41 or equivalent rotor, 18 C
7.	dissolve pellet (soft pipeting) in 1 x proteinase K buffer
	digest according to appr. protocol (Sambrook !). Generally with
	50 ug/ml pro reaction of 400 ul; 56C for 1 hour or 37 O/N
8.	add 1 ml chloroform, vortex 30 seconds.
9.	spin 10', big interphase.
10.	extract chloroform phase once, combine watery phases.
11.	extract with 2 x with phenol / chloroform, once with chloroform
12.	precipitate with one volume isopropanol, wash and dissolve in 50 
	to 100 ul H2O, test 5 ul on gel

if you need a lot of material, this protocol should be easy to scale up.
 
cheers,
clemens
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Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10,        D-50829 Koeln, Germany
Tel.xx49.221.5062-221    Fax.-213      e-mail: suter at vax.mpiz-koeln.mpg.d400.de
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