genome sequencing of intracellular bacterium (View forum version)


Posted 17 December 2013 - 01:04 AM

Dear all,

we are planning to sequence genome of intracelular bacterium, grown in a cell culture. All protocols I've read describe first to purify bacteria from the cell culture, and next to purify the genomic DNA from bacteria. Does anyone know the simplest technique to purify only bacteria from cell culture? I guess that for genomic DNA extraction from bacteria any commercial kit is OK. Eucariontic DNA contamination needs to be as low as possible. They say the best way to check contamination is with 18S rRNA real-time RT-PCR. Is this OK? How do I check the quality of purified DNA?

I would be very grateful any tip or advice given.

Best regrads to all!!!



Posted 17 December 2013 - 12:45 PM

The best way to isolate will be a gradient centrifugation method, which will probably also give you mitochondria and perhaps ER and golgi., but these may be removable with further purification steps.


18sRNA shouldn't be present in bacteria IIRC, so it should be good to use.


Quality of DNA can be estimated by running on a gel and looking for smearing of the DNA.