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restriction digestion


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#1 annc

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Posted 28 July 2010 - 09:36 PM

please answer.... i am new in this field
how to avoid some bands appear in the smear during restriction digestion of genomic DNA. is it because of any contamination in DNA or we have to change the RE

#2 HomeBrew

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Posted 29 July 2010 - 02:46 AM

Digestion of chromosomal DNA with a frequently-cutting restriction enzyme produces a series of bands differing from one another by small variations in size -- a smear. If you have a few brightly lit bands that stand out from the smear, it's because fragments of that size are present in the population of fragments more frequently than are others, and thus fluoresce more brightly (the degree of fluorescence is proportional to the molar concentration of the fragment).

Changing the restriction enzyme used will likely eliminate the brightly lit bands you're seeing, but may produce other brightly lit bands in a different pattern. The other possibility is that your genomic DNA includes a plasmid resident in whatever species you're recovering your DNA from (assuming it's bacterial). Plasmids are present in a higher copy number per cell than is the chromosome, thus there will be more copies of this DNA, and it will appear as more brightly fluorescing bands for the same reason as described above.

#3 annc

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Posted 29 July 2010 - 10:37 PM

Digestion of chromosomal DNA with a frequently-cutting restriction enzyme produces a series of bands differing from one another by small variations in size -- a smear. If you have a few brightly lit bands that stand out from the smear, it's because fragments of that size are present in the population of fragments more frequently than are others, and thus fluoresce more brightly (the degree of fluorescence is proportional to the molar concentration of the fragment).

Changing the restriction enzyme used will likely eliminate the brightly lit bands you're seeing, but may produce other brightly lit bands in a different pattern. The other possibility is that your genomic DNA includes a plasmid resident in whatever species you're recovering your DNA from (assuming it's bacterial). Plasmids are present in a higher copy number per cell than is the chromosome, thus there will be more copies of this DNA, and it will appear as more brightly fluorescing bands for the same reason as described above.



#4 annc

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Posted 29 July 2010 - 10:42 PM

thank you for your suggestion. but i am using plant genomic DNA.And for the further procedures they are telling continuous smear is needed.( glenn et al2005) . And also whether can we use the topo XL kit for frequently cutted genomic DNA? please answer.....

Edited by annc, 29 July 2010 - 10:43 PM.


#5 HomeBrew

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Posted 30 July 2010 - 03:53 AM

I don't have the reference, but by "continuous smear" do they mean a smear that runs from the top of your gel to the bottom (as an indicator that the DNA is fully digested), or do they imply that it also has to be a smear with equal band intensity throughout?


"Equal intensity throughout" is going to be hard to achieve -- there are likely always going to be some fragment sizes that are over-represented, and thus fluoresce more brightly, no matter what restriction enzyme you use. If, for example, your genome contains multiple copies of a transposon, or has sections of repetitive DNA, fragments derived from these are going to be present in multiples and be brighter, or perhaps just by chance there are several restriction fragments produced by digestion that are the "same size" (within the resolving cababilities of the gel) and fluoresce more brightly.


The TOPO XL kit is a PCR cloning kit. It is designed to clone PCR products by taking advantage of TA overhangs generated during PCR amplification of DNA. You're not generating PCR fragments, you're generating fragments by restriction digestion, thus a TA cloning vector would not be useful.

#6 annc

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Posted 30 July 2010 - 09:39 PM

thank you homebrew. i will tell you what is it... it is a paper for isolation of microatellites.what i understood by continuous smear is not in the same intensity from top to bottom. what they are telling is more intensity should be there in 500-1000bp range so that could use topo TA cloning kit. And ofcourse we are doing a PCR before cloning using the enriched DNA. And continuous i thought no bands. And if there is bands what will be the problem? And this TOPO Xl kit they are telling it is for large insert form 3 Kb - 10 Kb. so can we use it for below 3 kb. because it is already is there with me for some other work.
thanking you... expecting the reply....

#7 HomeBrew

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Posted 30 July 2010 - 09:56 PM

It sounds OK, but can you post a link to the reference so I can be sure that I understand correctly?

#8 annc

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Posted 02 August 2010 - 01:36 AM

sorry for delay.. i am giving the link here. or just u can give glenn &schable2005 in google and u will get it. My link

thanks in advance..




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