During the process of ecoli transformation by rDNA technology there is a chance of obtaining different ligation products like (gene of insert+gene of insert),(gene of insert+vector),(vector+vector),(gene of interest in opposite direction+vector)...how can we lessen these unwanted products?
Dear Neela,
THe question deserves appreciation. gene+vector is desired; so don't bother. improper orientation of gene results majorly when TOPO cloning is used; Other improper ligations could be avoided by the use of SIP or CIP, prior to ligation. Additionally, you would have a tag to purify your protein in the case of E. coli expressions (commercially available plasmids). So, just concentrate on the product of interest. During subsequent purification steps, such impurities would majorly be removed.
Hi Neela,
you can always play with the inset:vector ratio, anyway restriction analysis will be required to confirm the desired sizes. Why not to try to design and synthesize both your insert and vector de novo, also try other assembly methods: golden gate cloning,gibson assembly, etc.
@Neela Vineeth Teja Besides, the points above please use different restriction sites for the 5' and 3' end. When using the same sites getting two copies of the gene of interest ligated is a possibility albeit with lower probability.
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