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Chazin Lab Protocol for DNA Sequencing

notes

Written (6-2-04) by Jonathan Sheehan

Purpose

Let's say you've just done some subcloning or mutagenesis, and you want to confirm that the resultant plasmid has the sequence you want. Here are the steps:

Steps:

  1. Miniprep your plasmid using Qiagen's kit. This should give you 50ul at roughly 500ng/ul

  2. Check the concentration on the UV/vis spectrophotometer. A(260) * 50 = ng/ul. You can also check for purity [protein contamination] via A(260)/A(280)

  3. Dilute a sample for submission to the DNA sequencing core. They request 15ul at 25ng/ul in a regular eppendorf. Label the tube with a seven-character (or less) sample name and "xyzWJC", where xyz are your initials.

  4. If you're not using a T7 vector, you may have to provide primers, too. Check their form.

  5. Fill out the RTF file called 'master.rtf'. It would be best to use the latest version, which should be available at their website (www.seq.mc.vanderbilt.edu/DNA) (right now, it seems to be down). Use your grant number, (e.g. 4.04.253.0071 for CaBP grant) and <3Kb size

  6. Email the form to dna.sequence@vanderbilt.edu.

  7. Put your sample(s) in the fridge in the core lab (531 Light Hall). They should go in the front row of the Chazin box.

Results:

  1. You should get an email with the sequences (as text files) in 3-5 business days.

  2. Electropherograms are available as hardcopy at the core, and by ftp over the network. They contain more information than just the sequence.

  3. Analyze on Mac in SB core lab.

  4. First, generate a text file with a comparison sequence (e.g., the unmutated DNA, or the sequence you were hoping to generate)

  5. FTP: 160.129.208.184 (use fetch or terminal window; in theory, explorer can do this, but I haven't gotten it to work, yet) uname: chazin_w pwd: rpa70ab

  6. Unzip files with Unstuffit. This should give a .seq file (which you already got in the email... it's just text) and a .ab1 file (this is the raw data).

  7. Files (may not?) need to be converted from PC to Mac using utility in ConfProgFolder on Lab's desktop.

  8. Run Sequencher (icon is in the dock)

  9. Open new project

  10. Import sequence files: pick a result file (ab1 file), and also your comparison file.

  11. Click "automatically assemble" and it will align them into one contig.

  12. Click "overview" to see where your desired sequence falls in the sequenced area.

  13. Click "bases" to check individual ambiguities (marked with +) or differences (marked with a dot). After you select them, you can click "show chromatogram", and then edit the sequence (top row of letters) if the answers are clear.

  14. Click "summary" to get a report in printable form. Nice Options: "matching bases as dashes", "translations for fragments", View: "e.g. translate frame #1", Ruler: "courier6, squashed vertically and horizontally), right margin at 7.5 inches

  15. Save project.

  16. Print to mono1 for notebook (color doesn't work from this mac).