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Cloning Large Fragments in a pool of DNA fragments - (Mar/01/2005 )


In my experiment, genomic DNA was digested into fragments with different sizes from ~200-1000bp and then amplified by PCR. After that, I cloned the pool of fragments into a vector by TA cloning (TOPO2.1) and then the clones were sequenced. I found that most of the fragments cloned into the vector were small in size, about 200-300bp. I want to ask how to improve the experiment, so the large fragments, >300bp can be cloned. I know that I can add more vector or use some gel filtration column to eliminate the small fragments. However, are there any other methods? or have you faced this problem?


Run the mixture on a low melting agarose gel and extract the bands of required size and use them for cloning.