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Question about CsCl gradient purify chloroplast DNA!!!!

Question about CsCl gradient purify chloroplast DNA!!!! - Protocols and Methods Forum

Question about CsCl gradient purify chloroplast DNA!!!! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-21-2003, 12:22 PM
Mr.Q
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Default Question about CsCl gradient purify chloroplast DNA!!!!



Dear everyone,

I would like to prepare chloroplast DNA by using CsCl gradient.But I
still don't know how to prepare the CsCl gradient and how much CsCl
should I add in my chloroplast solution?Besides,how do I design the
centrifuge speed properly?The chloroplast DNA size is about
150Kb-170Kb.I will appriciate that anyone could tell me how to
prepare.

Thanks,


hsien chia
email:[Only registered users see links. ]
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  #2  
Old 10-21-2003, 01:01 PM
Duncan Clark
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Default Question about CsCl gradient purify chloroplast DNA!!!!

Historians believe that in newspost
<[Only registered users see links. ] > on Tue, 21 Oct 2003,
Mr.Q <[Only registered users see links. ]> penned the following literary
masterpiece:

Standard amounts of CsCl for separating plasmid from Xsomal is around
1g/ml with EtBr. At that lvel DNA should be around the middle of the
gradient formed during centrifugation.


Basically as high as it will go overnight on a vertical rotor or a
weekend for a normal rotor. I have a figure from years ago of 200,000g
for 17hours on a vertical.


Size is irrelevant, it's the buoyant density of the DNA that matters and
that will vary according to G/C ratio.


The original Maniatis has details of plasmid CsCl preps. Same principal
is involved.
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #3  
Old 10-22-2003, 02:25 AM
Eric R. Hugo
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Default Question about CsCl gradient purify chloroplast DNA!!!!

Duncan Clark <junk@[127.0.0.1]> wrote in news:nZViw+F+4Sl$EA5T@[127.0.0.1]:


Instead of Ethidium you might want to use bisbenzimide (Hoescht 33258 (I
think)). This causes separation based somewhat on AT content (primarily AT
runs). This might be needed to insure separation of the cpDNA from the
mtDNA that is probably present in your preparations.
Eric
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