I cloned my gene (474 bp) in the pCAMBIA plant expression vector and transformed in DH5 alpha strain. After colony PCR I took the positive colonies for plasmid isolation and kept the isolated plasmid at -20 DC temp.
But after one week when I again amplified the plasmid and positive colonies with gene-specific primers, it came back negative.
What could be the probable reason behind it? How can all positive colonies become negative? I rechecked it many times with a positive control but also I did not get any amplification.
maybe something went wrong with your plasmid isolation? Did you check the concentration and 260/280 ratios?
Hi Sweta,
In your last sentence, did you mean the positive control showed a band, but the plasmids isolated from the 'positive' colonies did not show any bands?
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