I have to clone a maize gene with pMAL and YFP vector. Now, I am doing TA cloning of that maize gene and planning to to subcloning with pMAL and YFP vector but i always get multiple bands after plasmid DNA extraction and failed to get complete sequencing result? What may be the reason? Can anybody suggest me to sucess cloning?
the reason you get multiple bands after cloning is plasmids might be two or confirmation, one with out gene, second with gene and third super coiled or so... now what u can do .. when u run plasmid after cloning then next well add ur amplcon or pcr product which you cloned and also TA plasmid without gene and gel extract that plasmid which above in band size of both reference bands
may be you have more than one plasmid vector in the bacterial cells strain, with different sizes.
check this issue to assure
May this gene prodcut is toxic in Ecoli,Maybe you need use special designed host strain or use low copy vector like pSC to clone you gene.
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