Hi all!
I am trying to construct a genomic DNA library of a gram-positive bacterium. I have isolated the genomic DNA and now I have to chop off the DNA into small fragments for cloning into a vector. I wanted to know which method should I follow: Partial restriction digestion or sonication? If sonication, then what should be my methodology to obtain evenly-sized fragments?
Regards
Yasir
In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase.
The easiest way would be to do partial digest, probably with a frequent cutting enzyme that has compatible ends to an infrequent cutter. For example you can do a partial digest with Sau3A (or MboI if not methylated) and ligate to BamHI digested plasmid, or cut with TaqI and ligate to ClaI digested plasmid. There are other examples as well.
If you use sonication or shearing then you need to do something with the ends for your ligation, such as ligating to a linker sequence or blunt end ligating to your vector.
But are you sure you really need a library? If the genome is sequenced you can get any gene you want through PCR, or if not then you could quickly generate the genome sequence.
Genomic DNA fragments can be obtained through a variety of methods, including enzymatic digestion with restriction endonucleases. In this method, the DNA is cut at specific sites, producing fragments of various sizes. Additionally, DNA can be fragmented through sonication or mechanical shearing. Finally, DNA can be purchased from commercial suppliers in the form of plasmids or bacterial artificial chromosomes (BACs).
Yasir Bashir the fragments will all have the same sticky ends on both sides so that there is no way to specify orientation. In other words it will be half and half. But if your library is large enough then all genes are likely to be found in the library multiple times with varying lengths and varying orientations.
Feel free to write directly if you wish.
I assume you are making a library from an organism whose genome has not been sequenced? If you know which gene you are trying to clone and have the sequence from some close homologs, it might just be easier to make primers and amplify your gene of interest.
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