Yeast chromosomal EcoRI digests

Michael Hamilton mhamilto at gpu.srv.ualberta.ca
Thu Aug 31 17:51:48 EST 1995


bundock at rulsfb.leidenuniv.nl (paul bundock) wrote:

>I'm not sure if anybody else has this problem but I seem to be unable to 
>efficiently digest yeast chromosomal DNA with EcoRI.I'm using the 
>recommended buffer and a large digestion volume of 200ul.The DNA is 
>purified using a lyticase protocol with many phenol steps.Does anyone 
>have experience with this? Thanks, Paul Bundock.


Many phenol steps??  Try the protocols listed in the Current Protocols
Handbook (bigRedBook) or in the Methods in Enzymology issue on yeast
(vol 188, I think)  Neither require the use of phenol, and I
consistently get >50kb fragments, acceptable yields, and DNA which is
easy to cut, PCR, label, sequence.....  

An alternative method is:  
1.  centrifuge 1.5 mls of saturated culture.  remove supernatant, and
wash with H2O.  
2.  Add 100 ul of 3% SDS,TE.  resuspend pellet.
3.  Hold at room temperature for 15-30 min, mixing by inversion.
4.  Add 500 ul of TE, mix.  
5.  Extract once with 600 ul of phenol.  
6.  Transfer aqueous phase to new tube, add 500 ul of isopropanol.  
	DNA may not be visible.  I sometimes find that adding salt(NaCL) 
	or holding at 4 C helps to precipitate the DNA
7.  Centrifuge, wash with 70% EtOH, resuspend in acceptable vol.  

usually yields 1-2 ug, about 20 kb in average size, and cuttable,
PCR-able, etc.  Good for screening. 
Reference is somewhere in NAR, I think.  I can send it to anyone who
is interested.     

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                                              | And have your way with me.




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