MIT:Sequencing BioBrick DNA

Sequence Request and Sample Preperation

Purpose

In order to fully verify the validity of any parts or part intermediates created, the DNA must be sequenced. This is done by the BioPolymers lab in the Cancer center, not by us.

Materials

• Prepped plasmid for sequencing
• 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 Form
   • Write your name and e-mail, and Endy as the 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
   • Leave the other 2 columns blank, as well as the pricing information at the bottom of the sheet
   • Keep one sheet for your records, put one sheet in the Complete shelf of the lab filing cabinet (in 564d), and bring one sheet with your tubes to the biopolymers lab
2. Make a 12 uL 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
   • 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 very small ones) and write the number from the form 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, and Endy Lab.
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).

Verifying Sequence Data

this is a test