User:Anthony Salvagno/Notebook/Research/2009/02/17/E. coli Colony Isolation
So if you/I recall yesterday I began the growing process of plasmid inserted E. coli. That sentence is rather awkward... I inserted plasmid DNA into E. coli and we inoculated them overnight. I did 4 samples:
- Control - no plasmid insertion
- 25ng pDNA
- 50ng pDNA
- 100ng pDNA
The reason behind the four trials is because the competent bacterial cells were made over a year ago and we assumed they wouldn't work as well as a fresh batch. So we tried several different amounts and were hoping to get a good batch to work with from that.
Apparently, the cells were too competent. Even the least concentration had way too many cells. It was hard to grab individual colonies from the batch. The technique is:
- Keep everything very sterile.
- Combine LB media with something I don't remember the name of Anthromycin or something that sounds like that.
- Put cocktail into test tubes (we used 10 with 2mL in each tube)
- Using a pipette tip, pluck a single colony from the dish.
- Drop into a single tube and slosh around
- Repeat for all 10 tubes
- Incubate in 37C shaker overnight.
In later experiments we will need to have less colonies on a single dish. This is because each colony will represent a different genomic fragment and we will need to isolate each fragment (for sequencing and tethering). If we get more than one colony in a tube the results will be horribly flawed (but may be work around-able).