MIT:Sequencing BioBrick DNA

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* Prepped plasmid for sequencing
* Prepped plasmid for sequencing
**You may want to read the [[Optimizing Sample for Sequencing|notes]] from the Biopolymers lab for optimizing your prepped DNA
**You may want to read the [[Optimizing Sample for Sequencing|notes]] from the Biopolymers lab for optimizing your prepped DNA
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* VF2 and VR1 verification primers (10x dilution especially for sequencing reactions)
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* VF2 and/or VR1 verification primers (10x dilution especially for sequencing reactions)
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**We have stock solutions of these primers
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**For constructs greater than 1.5kb, you'll want more intermediate primers (can get ~800bp of reliable sequence per primer.)
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* Sterile DI water
* Sterile DI water
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* 3x Sequencing Request Form
 
===Methods===
===Methods===
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#Fill out the [http://web.mit.edu/biopolymers/www/request_form.html Sequencing Request Form]
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#Fill out the [http://dnalims.mit.edu/index.html online submission form]
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#*Write your name and e-mail, and the name of your lab
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#*Print one sheet for your records, and bring one sheet with your tubes to the biopolymers lab.
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#*Give the Template ID as whatever you want (probably the part number)
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#Make a 12 μL mix as specified in the submission form.
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#*Primer ID is usually VF2 or VR1 (or an intermediate primer)
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#*From the 40μM stock solutions, you'll want 0.8μL of a 1:10 dilution of the stock.
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#*Vector is whatever vector your part is on
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#The sequencing request form is numbered. Put each mix into a PCR tube (if you have >4 samples, the sequencing center requests the use of PCR strips) and write the sample number from the form on the top of the tube.
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#*Leave the other 2 columns blank, as well as the pricing information at the bottom of the sheet
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#*Keep one sheet for your records, and bring one sheet with your tubes to the biopolymers lab.
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#**For the Endy lab, remember to leave a copy in the Complete shelf of the lab filing cabinet (in 564d).  
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#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).
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#*For the stock solutions, you'll want one microliter of primer (they have been pre-diluted so this works)
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#*The sequencing request form is numbered. Put each mix into a PCR tube (if you have >4 samples, the sequencing center requests the use of PCR strips) 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.
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#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.
#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.
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#*The Biopolymers lab is room [http://whereis.mit.edu/map-jpg?mapterms=E17-415&mapsearch=go 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.
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#*The Biopolymers lab is room [http://whereis.mit.edu/map-jpg?mapterms=E17-415&mapsearch=go 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.  
 +
#When you arrive put all the small PCR tubes into a large falcon tube (there is a stash of used falcons next to the small fridge). Then label the side of the large falcon with your name, the date, and Knight Lab and the order number.
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#Now you get to wait for your sequencing results to come back (usually 3-4 days).  You should be notified by email when they are available.  They are trying to reduce their turnaround time to 1 day. 
 +
 
 +
Note that the Biopolymers lab is generally only open during normal business hours, Mon-Fri, 9-5.
==Retrieve sequence data==
==Retrieve sequence data==
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[http://web.mit.edu/biopolymers/www/ftp_server_instructions.html Instructions] for retrieving sequence data.   
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[http://dnalims.mit.edu/index.html Login to dnalims] to retrieve sequence data.   
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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.
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==Verify sequence data==
==Verify sequence data==
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There are also various programs that the sequencing center recommends listed [http://web.mit.edu/biopolymers/www/dna_sequencing.html here].
There are also various programs that the sequencing center recommends listed [http://web.mit.edu/biopolymers/www/dna_sequencing.html here].
 +
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The dnalims online software also permits sequence analysis.  According to the sequencing center, it is quite good.
[[Category: Protocol]]
[[Category: Protocol]]

Revision as of 12:36, 15 May 2006

Back to Sequencing DNA

Contents

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/or VR1 verification primers (10x dilution especially for sequencing reactions)
  • Sterile DI water

Methods

  1. Fill out the online submission form
    • Print one sheet for your records, and bring one sheet with your tubes to the biopolymers lab.
  2. Make a 12 μL mix as specified in the submission form.
    • From the 40μM stock solutions, you'll want 0.8μL of a 1:10 dilution of the stock.
  3. The sequencing request form is numbered. Put each mix into a PCR tube (if you have >4 samples, the sequencing center requests the use of PCR strips) and write the sample number from the form on the top of the tube.
  4. 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.
  5. When you arrive put all the small PCR tubes into a large falcon tube (there is a stash of used falcons next to the small fridge). Then label the side of the large falcon with your name, the date, and Knight Lab and the order number.
  6. Now you get to wait for your sequencing results to come back (usually 3-4 days). You should be notified by email when they are available. They are trying to reduce their turnaround time to 1 day.

Note that the Biopolymers lab is generally only open during normal business hours, Mon-Fri, 9-5.

Retrieve sequence data

Login to dnalims to retrieve sequence data.

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.

The dnalims online software also permits sequence analysis. According to the sequencing center, it is quite good.

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