I've done cloning from PCR products and wanted to purify the plasmid. My miniprep protocol requires me to incubate transformed E coli in LB broth for 12 hours before processing. I did. In the morning when i wanted to retrieve the overnight culture, i found that the incubater stopped shaking and there were a clump of whitish thing (E coli?) in all tubes. I've got no idea when did it stop.However, i took those samples and proceeded with miniprep. After miniprep, i ran a 1h 1% TBE gel (without treatment with RE 1st) for my plasmid and found no band. Was it due to no shaking during incubation? Or something went wrong in miniprep? Or i shouldn't run a gel for plasmid without treatment with RE? Is it a routine to use RE to check the plasmid before sending to sequence?
There is a thread in this forum where ppl mentioned that they didn't purify the plasmid after sequencing. If i dont do miniprep, in what medium should i transfer my single colony? Does it need extra treatment before sequencing?
When doing cloning, why should we put the bacteria in a incubater with shaking function? Was it to evenly distribute the bacteria all over the culture? So that they will have enough nutrients? Then why some protocols require higher speed and some, lower?
Edited by hianghao, 08 September 2010 - 12:17 AM.