I have to proceed for digestion where the plasmid yield is not sufficient.
1.) Is the right amount of antibiotic that you use for seletion present in the growth medium? Has the antibiotic been stored correctly? Is it maybe an old batch?
2.) Do you use a kit for preparation of plasmid DNA? If yes, did you add ethanol to the washing solution (otherwise you will lose most of your plasmid DNA when you wash the column-bound DNA).
3.) Do you use an EDTA-buffered solution for final resuspension/elution? (otherwise DNAses can be active and degrade your plasmid DNA).
4.) Has the lysis step been performed for too long? Then it might be possible that you get clogging of the column by genomic DNA.
I hope these suggestions help you a bit.
Best regards,
Christian
yes kanamycin is the antibiotic i am using at 50mg/lit. i am using kit.initially the yield was around 300ng/ul. now it is hardly 2-5ng/ul. Yes ethanol is added to washing solution..lysis step is for 1 minute.....and elution buffer is TE...
may be your plasmid of interest is getting intergarted in to the host genome (bacterial DNA). please change bacterial strain
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