Preparing Sanger Sequencing (Eurofins)

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Only PCR product or Plasmid

Used if Eurofins will be added a primer to the Sanger sequencing tube.

  • Add 10ul of DNA to a 1.5ml microcentrifuge tube. The concentration should be:

PCR Products, Purified 0.100–0.300kb 10–20 ng/ul

PCR Products, Purified 0.301–1.000kb 20–40 ng/ul

PCR Products, Purified >1.000kb 40–60 ng/ul

Plasmid, Prepared < 1 .000kb 30-60 ng/ul

Plasmid, Prepared 1.0 << 6.0kb 60-150 ng/ul

Plasmid, Prepared 6.0 << 20.0kb 150-250 ng/ul

However, don't sweat it if you have less than this concentration. Please dilute back if your concentration is larger than the high end of these values.

  • Place a separate sequencing barcode on each tube, and send Eric the barcode letters/numbers, as well as the contents of the sample or samples. Each sample gets a different barcode.
  • Put a thin bit of parafilm around to the tube, to prevent it from opening.
  • Place all tubes in a small bag (see the top drawer, with the autoclave tape) and place in UPS shipping envelope (found in the bookcase in the write-up area).
  • Have Eric print the order slip (which goes into the envelope) as well as the shipping label. The shipping label gets cut out and taped on all 4 sides to the UPS envelope.
  • Once it is ready, put in the shipping mailbox behind the dining center, by the blue bus stop. It gets collected by ~5:30pm-6:00pm each day; keep samples in the lab refrigerator or -20 freezer if they are ready after this time.

Plasmid + primer, or PCR + primer

Same procedure, but use 5ul DNA. Add 5ul of a single primer at the same concentration as PCR -- a 1:10 dilution of the blue-capped, stock solution.

Key Primers

  • If sequencing a P-80 derived CRISPR plasmid, use primer O-263
  • If sequencing a P-52, pET28a(+) derived expression plasmid, use the T7 forward primer, provided by Eurofins.