Colony hyb with Bacillus

Robert Coelen robert at arbo.microbiol.uwa.oz.au
Fri Jul 24 03:13:51 EST 1992


In article <qzgmkh- at lynx.unm.edu> kim at m44.unm.edu writes:
>In article <1992Jul23.025622.9263 at menudo.uh.edu>, bchs1b at Elroy.UH.EDU (Michael Benedik) writes:
>>In article <1992Jul21.154049.25431 at usenet.ins.cwru.edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>
>
><Stuff about ss phagemid DNA preps omitted>
>
>Instead of using gradient centrifugation or PEG, there is a method that uses
>Glacial Acetic Acid and Sodium Perchlorate.  It is a glass-binding type DNA
>purification, like Geneclean.  I have never tried it, but the reference is:
>
>Nucleic Acids Research 15:5507-5516
>
>A modification of this method was posted a while ago as:
>
>1. In a 1.5 ml tube, mix 1 ml cleared phagemid supernatant with 10 microliters
>of glacial acetic acid.  Sit r.t. for 5 minutes to precipitate phage.
>
>2.  Spin out the phage pellet.  Drain.
>
>3.  Resuspend in 4.5 M Sodium Perchlorate (I didn't get the volume to use),
>made up in TE buffer.  Sit 50 C for 10 minutes.
>
>4.  Add 5 microliters glassmilk from Geneclean or home-made.  Rock on ice 10
>minutes.  (So the volume to resuspend should be maybe 100 - 300 microliters, I
>would guess).
>
>5.  Spin out the glass.  Drain.  Do NEW wash 3 times.
>
>6.  Thoroughly drain the glass pellet, and elute the ss DNA in 50 microliters
>water.
>
>
>If you try this, post a report on how it works/doesn't work.  I'd like to know.
>
>Daniel Kim
>KIM at FLOVAX.LANL.GOV


Lee Sammels in my lab cooked up a method that runs something like the
above and involves glass milk and the sequence we get from the ssDNA is
just BEAUTIFUL !!!!

Here are the salient details:

1.  Do a standard PEG/NaCl ppt of the phage from 1.5 ml of medium
2.  Discard supernatant
3.  Resuspend phage in 50 ul of 4.5M NaClO4 and combine 4 50 ul lots into
    one 200 ul lot
4.  Leave this with occasional vortexing at RT for 20'
5.  Add 10 ul of glass milk suspension and leave at RT for 15', vortex
    occasionally
6.  Spin the glass out, wash 3x with 50% etoh in TE
7.  Resuspend ssDNA in 20 ul of your favourite liquid
8.  Enjoy your sequence !!

This method is not yet optimised, but we'll get to that and repost. 


Robert Coelen
Dept of Microbiology
University of Western Australia



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