Submitting sequencing
- Submit 10 uL of miniprep plasmid
- And 3 uL of sequencing primer per read
- Different services want different volumes, premixing and submission methods
- Next day you get two files: the trace (peaks) and the read (GenBank)
Step by step: Cycle Sequencing tutorial · bench cheatsheet
Always read the trace, not just the sequence. The base calls at the two ends of a read are guesses, made where the peaks are not resolved well enough to call anything. And a clean double peak in the middle of an otherwise good read is worth knowing how to read: E. coli is haploid and your colony is a clone, so there is no such thing as a heterozygote here. Two bases at one position means two templates, which almost always means you picked two colonies at once. What you send and what comes back: ten microlitres of miniprep and three of primer per read, and next day a trace of coloured peaks and a sequence file of base calls.