Sequencing BioBrick DNA-PDF

Purpose

To confirm the physical DNA resulting from a BioBrick standard assembly step has the same sequence as that part in the registry, the physical DNA must be sequenced. This is done by the BioPolymers lab in the Cancer center.

Sequence Request and Sample Preparation

Materials

• Prepped plasmid for sequencing
  • You may want to read the notes from the Biopolymers lab for optimizing your prepped DNA
• VF2 and VR1 verification primers (10x dilution especially for sequencing reactions)
  • We have stock solutions of these primers
  • For constructs greater than 1.5kb, you’ll want more intermediate primers (can get ~800bp of reliable sequence per primer.)
• Sterile DI water
• 3x Sequencing Request Form

Methods

1. Fill out the Sequencing Request WebForm
   • Write your name and e-mail, and the name of your lab
   • Give the Template ID as whatever you want (probably the part number)
   • Primer ID is usually VF2 or VR1 (or an intermediate primer)
   • Vector is whatever vector your part is on
   • Print 2 copies, bring one with your tubes to the biopolymers lab.
     • For the Endy lab, remember to leave a copy in the Complete shelf of the lab filing cabinet (in 564d).
2. Make a 12 μL mix as specified in the top row of the Form’s Sample Prerequisite table. Basically there will be 2x the number of tubes as there are parts being sequenced (one with VF2 and one with VR1).
   • For the stock solutions, you’ll want one microliter of primer (they have been pre-diluted so this works)
   • The sequencing request form is numbered. Put each mix into a PCR tube (the sequencing center requests the use of PCR strips) and write the number from the webform on the top of the tube. Put all the small PCR tubes into a large falcon tube. Then label the side of the large eppendorf with your name, the date, Endy Lab, and the order number given by the webform.
3. Deliver all the tubes and the form to the Bio Polymers lab in the Cancer Center. Go to the Cancer center, take the elevator to the 4th floor, take a right, it will be your 2nd-ish door on the left.
   • The Biopolymers lab is room E17-415. Inside, there is a brown fridge with a clear plastic thing attached to the front. Put your request from in the clear plastic thing and your large eppendorf tube inside the fridge. Now you get to wait for your sequencing results to come back (usually 3-4 days). Note that the Biopolymers lab is generally only open during normal business hours, Mon-Fri, 9-5.

Retrieve sequence data

Instructions for retrieving sequence data.

If you are unable to connect to the ftp server and it is mid-morning or so, then wait 10 minutes and try again. It appears as if (but has not been verified) that the Biopolymers ftp server doesn’t permit incoming connections while they are uploading sequence data to their server.

Verify sequence data

One option is to use VectorNTI to align your expected sequences with the .abi data files generated by the sequencing center via the ContigExpress module.

The registry permits blasting against the parts database with the option of only blasting against basic parts. This can be a quick way to determine whether your sequence is right or not.

There are also various programs that the sequencing center recommends listed here.