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genomic DNA quality for genome walking


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#1 zx0819

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Posted 13 September 2009 - 06:57 PM

I am new in the area of genome walking and I am going to do it for the first time in the next few months. In order to make a good start, the most important thing is to extract the genomic DNA from my interesting plant. I want to ask the people here that is there any special requirement for the genomic DNA quality? I am using Qiagen DNeasy Mini Plant Kit to extract the genomic DNA before. I do not know if it is suitable the the downstream application of genome walking.

I will use the universal genome walker kit from clontech to isolate the promoter of my interested gene. In the manual, it mentioned the quality of genomic DNA: it should be bigger than 50 kb with minimum smearing. However, my genomic DNA seems not meet this requirement after checking on the agarose gel. So can anyone here who did genome walking before give me some suggestions on which kit or protocol you are using for the subsequent digestion library construction?

Thanks very much in advance!

#2 gleb.kudr

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Posted 16 September 2009 - 05:36 AM

I will use the universal genome walker kit from clontech to isolate the promoter of my interested gene. In the manual, it mentioned the quality of genomic DNA: it should be bigger than 50 kb with minimum smearing. However, my genomic DNA seems not meet this requirement after checking on the agarose gel. So can anyone here who did genome walking before give me some suggestions on which kit or protocol you are using for the subsequent digestion library construction?

Thanks very much in advance!


It is good enough. I think you can't possibly obtain a 50kb resolution on the basic agarose gel so relax and enjoy :(

Also the simple CTAB protocol works well.

#3 zx0819

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Posted 26 September 2009 - 11:52 PM

I will use the universal genome walker kit from clontech to isolate the promoter of my interested gene. In the manual, it mentioned the quality of genomic DNA: it should be bigger than 50 kb with minimum smearing. However, my genomic DNA seems not meet this requirement after checking on the agarose gel. So can anyone here who did genome walking before give me some suggestions on which kit or protocol you are using for the subsequent digestion library construction?

Thanks very much in advance!


It is good enough. I think you can't possibly obtain a 50kb resolution on the basic agarose gel so relax and enjoy :D

Also the simple CTAB protocol works well.

Thanks. Is there any requirement on DNA concentration? Generally, Mini Kit can extract only 10-30 mg total DNA in 100 ul elution buffer.




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