Reshma P. Shetty at 18:13, 2 January 2007

Reshma P. Shetty — Tue, 02 Jan 2007 18:13:42 GMT

←Older revision		Revision as of 18:13, 2 January 2007
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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.

-	==~~Sequence Request and Sample Preparation~~==	+	==Materials==

-	~~===Materials===~~
-
-	* Prepped plasmid for sequencing
-	**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
		+	* Prepped plasmid for sequencing
		+	**You may want to read the [[Optimizing Sample for Sequencing\|notes]] from the Biopolymers lab for optimizing your prepped DNA
		+
		+	{\|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.-->
		+	\| style="background:#f0f0f0;" \| '''Template'''
		+	\| style="background:#f0f0f0;" \| '''Quantity'''
		+	\|-
		+	\| colspan="2"\| ''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
		+	\|}

-	===~~Methods~~===	+	===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]]
		+	[[Category: In vitro]]
		+	[[Category: DNA]]

Reshma P. Shetty at 17:36, 15 May 2006

Reshma P. Shetty — Mon, 15 May 2006 17:36:48 GMT

New page

''Back to [[Sequencing DNA]]''

==Purpose==

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.

==Sequence Request and Sample Preparation==

===Materials===

* Prepped plasmid for sequencing
**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)
* Sterile DI water

===Methods===

#Fill out the [http://dnalims.mit.edu/index.html online submission form]
#*Print one sheet for your records, and bring one sheet with your tubes to the biopolymers lab.
#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.
#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.
#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 [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.
#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==

[http://dnalims.mit.edu/index.html 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 [http://parts.mit.edu/r/parts/partsdb/blast.cgi 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 [http://web.mit.edu/biopolymers/www/dna_sequencing.html here].

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

[[Category: Protocol]]

Knight:Sequencing DNA - Revision history

Reshma P. Shetty at 18:13, 2 January 2007

Reshma P. Shetty at 17:36, 15 May 2006