CsCl plasmid RNA?
hamel at ccu.umanitoba.ca
Thu Dec 2 15:45:02 EST 1993
CsCl-EtBr banded plasmids will always contain concomitant RNAs. This has
been well documented (I'd dig through my files if need be).
Simple test of this is to treat equal aliquots (eg. 5 ug) of final purified
plasmid prep into 50 uL TE with versus without 0.2 N NaOH -> 37*C/15 min ->
neutr with 6 uL 3 M NaAc (pH 4-5) -> add 200 uL 100% EtOH, mix -> sit
overnite -20*C to +4*C -> microfuge, wash, dry -> dissolve each pellet in
same vol. H2O -> determine A230/A260/A280 for each AND check same volume
aliquot of each on mini gel ... Abs will be lower for NaOH treated prep BUT
plasmid band intensities should be same ... due to alk. hydrolysis of RNA,
Abs is lower for NaOH treated ... thus reason why alk. den. is used for
preparing ds plasmid for sequencing.
Also, PEG ppt'n PRIOR to CsCl-EtBr ultracent. will aid in reducing
concomitant RNA problems ... simply isprop ppt after NaOH-SDS/NaAc neutr
-> dissolve pellet in TE (30 mL for 1 L cells worth) -> add 5% PEG/8,000
and 0.5 M NaCl (final conc.) -> mix, sit on ice/ON -> harvest pellet ->
dissolve pellet in TE -> add 1.1 gm/mL CsCl/2 mg/mL EtBr -> on ice 1 hr ->
spin 10,000 xg/1/2 hr -> transfer to ultracent tube, avoiding protein-EtBr
pellicle scum along sides/bottom, which can otherwise interfere with
CsCl-EtBr ultracent band recovery -> supplement with more EtBr if need be,
judging from EtBr red color intensity (can add 1 more mL of 5 mg/mL stock
to 15 mL CsCl-EtBr-plasmid).
Andre Hamel email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services lab tel.: (204) 945-7630
545 University Crescent, FAX:(204) 945-8062
CANADA R3T 5S6 ********************
In article <1993Dec2.143101.12805 at alw.nih.gov> Jim Owens <jow at helix.nih.gov> writes:
>In article <CHDoH7.Dv7 at fiu.edu> Derek M. Harkins, harkinsd at solix.fiu.edu
>>But then I re-read the initial question and the plasmid nucleic acids
>>have been spun through CsCL--therefore no contaminating RNA should be
>>to skew the readings of the spectrophotometer.
>I would not assume that there is no RNA to contaminate the plasmid unless
>there is an agorose gel to prove it.
>Years ago, when I routinely used CsCl gradients for plasmid preps, fixed
>angle rotors were used for the gradient centrifugations. RNA pellets
>under the conditions used and could be seen smeared along the side of the
>tubes away from the center of rotation. The ccc plasmid band would
>always be contaminated by RNA that came off the tube's wall. A second
>CsCl gradient was needed to eliminate the RNA.
>An RNaseA treatment before the centrifugation resulted in oligomers of
>RNA spread all over the gradient. The RNA oligomers in the plasmid band
>would not go away with a second centrifugation since their density
>matched that of the plasmid.
>Since so many people have trouble with fluorescent dyes in measuring DNA,
>and since you should have so much DNA from a CsCl gradient plasmid prep,
>uv absorbance is the method of choice in my humble opinion.
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