Genomic DNA cutting - (Jan/19/2007 )
i am new to this forum. i isolated genomic DNA from cancer cells and i digested that dna with hindIII and then i run gel . i swa smear . i didnt understand and i am very new to this one.how i can cut my desired band. please tell some tips if possible in detail.
It souds to me that you have lots!!! of DNA. trying to digest gDNA and see a sole band is not easy. Remember that you have all the genome and first you have to give plenty of time to the enzyme to find the sites and cut, so 1-3h asay will not function. for what you are digesting the DNA?if is for PCR then go ahead and try it, but if is for other type of asay then you have to dilute the DNA, then digest. If you explain more, will be easier for me give an answer.
thank you very much for your reply. actually i want to do sourthern blot after cutting the desired band of mine in the gel. can you tell me the protocol where i can found for digestion of genomic DNA
hi can any one tell how to analyze genomic dna using southern blot and i know before that i have to digest gdna . how to do that one i mean any useful tips to digest gDNA and what concentration of agarose i have to use and the agarose picture how it looks like . when i rum once i saw smear but i didnt saw any bands. please tell me that one can see bands in genomic DNA OR ONLY SMEAR or smear with bands. PLEASE GIVE ME DETAILED REPLY AS SOON AS POSSIBLE. pleaseeeeeeeeeeeeeeeeeeeeeeee
You could see bands and in the upper part of the gel a smear. The easiest way will be making a PCR, run in a gel and then make a southern of that gel.
i am really very thankful to you and actually i didnt saw any bands can you tell me what is the possible problem and you told me that to run a PCR . it could be very nice if u tell me how to run a PCR i mean programme and adding components . i am new to this technique thats why i am asking everything.
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are you seeing the smear in the gel or on the probed blot?
smear in the gel is normal. you should see a defined band on the blot after it has been probed for a specific sequence.
thank you for your reply. i am seeing smear in the gel , i get confused and i didnt blot the gel.
please give me a reply pleaseeeeeeeeeeeee