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Hello All -
I would like to have some advice concering a project I'm working on. We have a very nice CloneMiner cDNA library made from a eukaryotic microbe that we would like to sequence using 454 or Illumina. Getting new RNA for a standard RNAseq library will not be possible. The library is in bacterial stocks with a good titer and large average insert size (3 kb), we have sequenced 2000 clones by Sanger that looked really good, next to no rRNA contamination.
I'm hoping to purify plasmids from the stocks and prepare libraries using the Nextera kit from Epicentre, however I would not like to end up with to much empty vectors in the prep nor with to little plasmid to do anything useful. I don't know of any simple way of excising the library so we are prepared to get half the data as plasmid..
Does anyone now how many cell divisions the ccdB suicide gene needs to be active before those plasmids are lost. I would like to grow the cells the minimum amount of cell divisions not to risk to much uneven representation.
I would be grateful for any suggestions, tips or tricks.
/Jon
I would like to have some advice concering a project I'm working on. We have a very nice CloneMiner cDNA library made from a eukaryotic microbe that we would like to sequence using 454 or Illumina. Getting new RNA for a standard RNAseq library will not be possible. The library is in bacterial stocks with a good titer and large average insert size (3 kb), we have sequenced 2000 clones by Sanger that looked really good, next to no rRNA contamination.
I'm hoping to purify plasmids from the stocks and prepare libraries using the Nextera kit from Epicentre, however I would not like to end up with to much empty vectors in the prep nor with to little plasmid to do anything useful. I don't know of any simple way of excising the library so we are prepared to get half the data as plasmid..
Does anyone now how many cell divisions the ccdB suicide gene needs to be active before those plasmids are lost. I would like to grow the cells the minimum amount of cell divisions not to risk to much uneven representation.
I would be grateful for any suggestions, tips or tricks.
/Jon