help: genomic DNA extraction from tissue

s.pritchard mmd280 at sysa.abdn.ac.uk
Fri Jan 16 09:25:39 EST 1998


Z.Huang (huang.zhong at med.kuleuven.ac.be) wrote:
: Hi,

: I encountered a problem during genomic DNA extraction from human 
: intestine tissue. My brief protocol is as follows: 1. homogenize tissue 
: in TE buffer. 2.add SDS and proteinase K. incubation at 50c for half hour 
: followed by 37c for 1 hour or overnight. Now the mixture is very 
: viscouse. 3.Extract by saturated phenol (pH 8.0), repeat once time. 4. 
: Chloroform:Isoamyle alchole (1:24) extraction. 5. Absolute ethanol 
: precipitation. I can see viscouse phase after ethanol added. Then I 
: transfer the viscouse phase by "U" shaped pasteure pipette to a fresh 
: tube. It seems well done at this stage. 6. Air dry for a while, dissolve 
: with TE buffer. I can get A260/A280 ~1.9 and the yield is good. 

: **My PROBELM**: When I test the genomic DNA on 0.7% agarose gel I find a 
: lot of smearing from hundreds of bp to 24kb inspite I can see the desired 
: strong band of high molecular genomic DNA band (~23-24kb). It's highly 
: doubted that degradation or shearing of DNA happened during the 
: extraction process.(because I also tested another genomic DNA sample 
: prepared by Maniatis protocol and the sample just show one strong 23kb 
: band without any smearing). Can anyone out there show me a right way to 
: extract high molecular genomic DNA. I need it for further southern blot.

: Thank you very much for your help. Please also reply by e-mail: 
: han at biogate.com.

: Han
: K.U.Leuven

Sorry but it sounds to me like you are getting some degradation of you DNA.
How I dont really know could be mechanical are you shaking or vortexing
the prep after the proteinase K digestion? If so ....dont.
Try cutting the ends of your tips or if your loaded buy those genomic DNA 
tips that are wider bore.

Could the material you are extracting DNA from contain higher amount of DNase
(Unlikely)

Hope you get it sorted
Stuart



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