Knight:Sequencing DNA

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Current revision (13:13, 2 January 2007) (view source)
 
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To confirm the physical DNA resulting from a BioBrick standard assembly step has the same sequence as that part in the [http://parts.mit.edu registry], the physical DNA must be sequenced.  This is done by the [http://web.mit.edu/biopolymers/www/dna_sequencing.html BioPolymers lab] in the Cancer center.
To confirm the physical DNA resulting from a BioBrick standard assembly step has the same sequence as that part in the [http://parts.mit.edu registry], the physical DNA must be sequenced.  This is done by the [http://web.mit.edu/biopolymers/www/dna_sequencing.html BioPolymers lab] in the Cancer center.
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==Sequence Request and Sample Preparation==
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==Materials==
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===Materials===
 
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* Prepped plasmid for sequencing
 
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**You may want to read the [[Optimizing Sample for Sequencing|notes]] from the Biopolymers lab for optimizing your prepped DNA
 
* VF2 and/or VR1 verification primers (10x dilution especially for sequencing reactions)
* VF2 and/or VR1 verification primers (10x dilution especially for sequencing reactions)
* Sterile DI water
* Sterile DI water
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* Prepped plasmid for sequencing
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**You may want to read the [[Optimizing Sample for Sequencing|notes]] from the Biopolymers lab for optimizing your prepped DNA
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{|border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;" <!-- This line here formats your table for you.  Change the code to change the formatting of your table.-->
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| style="background:#f0f0f0;" | '''Template'''
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| style="background:#f0f0f0;" | '''Quantity'''
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|-
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| colspan="2"| ''PCR products''
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|-
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|&bull;  100-200 bp
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| 1-3 ng
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|-
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|&bull;  200-500 bp
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| 3-10 ng
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|-
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|&bull;  500-1000 bp
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| 5-20 ng
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|-
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|&bull;  1000-2000 bp
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| 10-40 ng
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|-
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|&bull;  >2000 bp
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| 40-100 ng
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|-
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|Single-stranded DNA
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| 50-100 ng
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|-
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|Double-stranded DNA
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| 200-500 ng
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|-
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|Large DNA BACs, PACs, YACs, cosmids, fosmids
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| 0.5-1.0 ug (use 5-10 pmoles of primer)
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|-
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|Bacterial genomic DNA
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| 2-3 ug (use 6-13 pmoles of primer)
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|- 
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|Primer (unless noted above)
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| 3.2 pmol
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|}
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===Methods===
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===Procedure===
#Fill out the [http://dnalims.mit.edu/index.html online submission form]
#Fill out the [http://dnalims.mit.edu/index.html online submission form]
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[[Category: Protocol]]
[[Category: Protocol]]
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[[Category: In vitro]]
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[[Category: DNA]]

Current revision

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.

Materials

  • VF2 and/or VR1 verification primers (10x dilution especially for sequencing reactions)
  • Sterile DI water
  • Prepped plasmid for sequencing
    • You may want to read the notes from the Biopolymers lab for optimizing your prepped DNA
Template Quantity
PCR products
• 100-200 bp 1-3 ng
• 200-500 bp 3-10 ng
• 500-1000 bp 5-20 ng
• 1000-2000 bp 10-40 ng
• >2000 bp 40-100 ng
Single-stranded DNA 50-100 ng
Double-stranded DNA 200-500 ng
Large DNA BACs, PACs, YACs, cosmids, fosmids 0.5-1.0 ug (use 5-10 pmoles of primer)
Bacterial genomic DNA 2-3 ug (use 6-13 pmoles of primer)
Primer (unless noted above) 3.2 pmol

Procedure

  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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