genomic dna isolation - (May/03/2011 )
Hi all
I have a simple query regarding genomic dna isolation. The eukaryotic genome is very large composing of several chromosomes of varied length and size , then why do we get a single genomic intact dna upon isolation and not more than one depending upon the chromosome number.
Thanks in advance.
You do not get intact eukaryotic DNA except in very rare circumstances, and certainly not with any normal gDNA prep technique. The DNA is extremely long and fragile, and will be broken by almost any manipulation. The DNA will indeed start out as chromosome sized fragments, with as many separate ones as there are chromosomes. But you'll never see it. An exception is small chromosomes of simple eukaryotic cells such as S. cereviciae (yeast) which has 200-1600 Kbp chromosomes, which can be separated and visualized with pulsed field gels. This takes using agarose embedded cells which are lysed in-place, and never manipulated in solution.
deespike on Tue May 3 12:57:32 2011 said:
Hi all
I have a simple query regarding genomic dna isolation. The eukaryotic genome is very large composing of several chromosomes of varied length and size , then why do we get a single genomic intact dna upon isolation and not more than one depending upon the chromosome number.
Thanks in advance.
You are not getting a single genomic intact DNA. What makes you think that you are getting single genomic intact DNA.
http://www.bioprotocols.info/index.php
phage434 on Wed May 4 02:37:47 2011 said:
You do not get intact eukaryotic DNA except in very rare circumstances, and certainly not with any normal gDNA prep technique. The DNA is extremely long and fragile, and will be broken by almost any manipulation. The DNA will indeed start out as chromosome sized fragments, with as many separate ones as there are chromosomes. But you'll never see it. An exception is small chromosomes of simple eukaryotic cells such as S. cereviciae (yeast) which has 200-1600 Kbp chromosomes, which can be separated and visualized with pulsed field gels. This takes using agarose embedded cells which are lysed in-place, and never manipulated in solution.
chromatin on Sat May 7 21:48:58 2011 said:
deespike on Tue May 3 12:57:32 2011 said:
Hi all
I have a simple query regarding genomic dna isolation. The eukaryotic genome is very large composing of several chromosomes of varied length and size , then why do we get a single genomic intact dna upon isolation and not more than one depending upon the chromosome number.
Thanks in advance.
You are not getting a single genomic intact DNA. What makes you think that you are getting single genomic intact DNA.
is it possible to get single genomic intact DNA if the DNA is bacterial DNA (around 5M)? i'm doing genomic DNA extraction for bacteria (phenol/chloroform extraction & kit extraction as well) and i have been trying for more than 2 months but all i get is smear when i run gel. what is the possible reasons?
Read my comments previous to this. You will never see a band for genomic DNA on an ordinary agarose gel. You are getting exactly what you should expect, a smear.
phage434 on Mon May 30 10:19:07 2011 said:
Read my comments previous to this. You will never see a band for genomic DNA on an ordinary agarose gel. You are getting exactly what you should expect, a smear.
when i searched online, those kits available in the market, could get a band for genomic DNA.
The attached pic shows the isolation of genomic DNA from both Gram Positive and Gram Negative bacteria using Norgen Bacterial Genomic DNA Isolation kit. they could get a band without smear. that's why i wonder why i couldn't get pic like these.
i would like to send my samples for whole genome re-sequencing. they want to see the intact band in gel.
