MIT:Sequencing BioBrick DNA - Revision history

Reshma P. Shetty: /* Purpose */

2008-01-07T20:22:46Z

Purpose

←Older revision		Revision as of 20:22, 7 January 2008
Line 3:		Line 3:
	==Purpose==		==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.	+	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/ BioPolymers lab] in the Cancer center.

	==Sequence Request and Sample Preparation==		==Sequence Request and Sample Preparation==

Reshma P. Shetty at 23:34, 9 May 2007

2007-05-09T23:34:16Z

←Older revision		Revision as of 23:34, 9 May 2007
Line 46:		Line 46:
	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].

-	[[Category: Protocol]]	+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:DNA]]

Jason R. Kelly: /* Methods */

2006-09-05T16:24:08Z

Methods

Reshma P. Shetty: reverted, edited the wrong page

2006-05-15T17:37:27Z

reverted, edited the wrong page

←Older revision		Revision as of 17:37, 15 May 2006
Line 11:		Line 11:
	* 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
-	* VF2 and~~/or~~ VR1 verification primers (10x dilution especially for sequencing reactions)	+	* 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		* Sterile DI water
		+	* 3x Sequencing Request Form

	===Methods===		===Methods===

-	#Fill out the [http://~~dnalims~~.mit.edu/~~index~~.html ~~online submission form~~]	+	#Fill out the [http://web.mit.edu/biopolymers/www/request_form.html Sequencing Request Form]
-	#*~~Print~~ one sheet for your records, and bring one sheet with your tubes to the biopolymers lab.	+	#*Write your name and e-mail, and the name of your lab
-	#Make a 12 μL mix as specified in the ~~submission form~~.	+	#*Give the Template ID as whatever you want (probably the part number)
-	#*~~From~~ the ~~40μM~~ stock solutions, you'll want ~~0.8μL~~ of ~~a 1:10 dilution of the stock.~~	+	#*Primer ID is usually VF2 or VR1 (or an intermediate primer)
-	#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.	+	#*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, and bring one sheet 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).
		+	#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 (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.
	#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.
-	#*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.	+	#*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.
-	#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==		==Retrieve sequence data==

-	[http://~~dnalims~~.mit.edu/~~index~~.html ~~Login to dnalims~~] ~~to retrieve~~ sequence data.	+	[http://web.mit.edu/biopolymers/www/ftp_server_instructions.html 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==		==Verify sequence data==
Line 39:		Line 46:

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

	[[Category: Protocol]]		[[Category: Protocol]]

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

2006-05-15T17:36:14Z

←Older revision		Revision as of 17:36, 15 May 2006
Line 11:		Line 11:
	* 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
-	* VF2 and VR1 verification primers (10x dilution especially for sequencing reactions)	+	* VF2 and/or 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		* Sterile DI water
-	* 3x Sequencing Request Form

	===Methods===		===Methods===

-	#Fill out the [http://~~web~~.mit.edu/~~biopolymers/www/request_form~~.html ~~Sequencing Request Form~~]	+	#Fill out the [http://dnalims.mit.edu/index.html online submission form]
-	#*~~Write your name and e-mail, and the name of your lab~~	+	#*Print one sheet for your records, and bring one sheet with your tubes to the biopolymers lab.
-	~~#*Give the Template ID as whatever you want (probably the part number)~~	+	#Make a 12 μL mix as specified in the submission form.
-	~~#*Primer ID is usually VF2 or VR1 (or an intermediate primer)~~	+	#*From the 40μM stock solutions, you'll want 0.8μL of a 1:10 dilution of the stock.
-	~~#*Vector is whatever vector your part is on~~	+	#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.
-	~~#*Leave the other 2 columns blank, as well as the pricing information at the bottom of the sheet~~	+
-	~~#*Keep~~ one sheet for your records, and bring one sheet 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)~~.	+
-	#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 (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~~.	+
	#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.
-	#*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.	+	#*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==		==Retrieve sequence data==

-	[http://~~web~~.mit.edu/~~biopolymers/www/ftp_server_instructions~~.html ~~Instructions~~] ~~for retrieving~~ sequence data.	+	[http://dnalims.mit.edu/index.html Login to dnalims] to retrieve 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==		==Verify sequence data==
Line 46:		Line 39:

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

	[[Category: Protocol]]		[[Category: Protocol]]

Hkeller at 00:44, 1 March 2006

2006-03-01T00:44:54Z

←Older revision		Revision as of 00:44, 1 March 2006
Line 31:		Line 31:
	#*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.		#*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.
	#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.
-	#*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).	+	#*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.

	==Retrieve sequence data==		==Retrieve sequence data==

Reshma P. Shetty: /* Retrieve sequence data */

2005-12-06T00:26:48Z

Retrieve sequence data

←Older revision		Revision as of 00:26, 6 December 2005
Line 35:		Line 35:
	==Retrieve sequence data==		==Retrieve sequence data==

-	[http://web.mit.edu/biopolymers/www/ftp_server_instructions.html Instructions] for retrieving sequence data.	+	[http://web.mit.edu/biopolymers/www/ftp_server_instructions.html 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==		==Verify sequence data==

Barry Canton at 14:54, 5 December 2005

2005-12-05T14:54:02Z

←Older revision		Revision as of 14:54, 5 December 2005
Line 1:		Line 1:
		+	''Back to [[Sequencing DNA]]''
		+
	==Purpose==		==Purpose==

Barry Canton at 22:30, 3 December 2005

2005-12-03T22:30:24Z

←Older revision		Revision as of 22:30, 3 December 2005
Line 1:		Line 1:
-	~~''This page contains information about sequencing BioBricks~~ parts. It is ~~written with some details specific to people working at MIT~~.''	+	==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==		==Sequence Request and Sample Preparation==
-
-	~~===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.

	===Materials===		===Materials===

Barry Canton: /* Purpose */

2005-12-03T22:14:44Z

Purpose

←Older revision		Revision as of 22:14, 3 December 2005
Line 5:		Line 5:
	===Purpose===		===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 [http://web.mit.edu/biopolymers/www/dna_sequencing.html BioPolymers lab] in the Cancer center~~, not by us~~.	+	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.

	===Materials===		===Materials===

←Older revision		Revision as of 16:24, 5 September 2006
Line 19:		Line 19:
	===Methods===		===Methods===

-	#Fill out the [http://~~web~~.mit.edu~~/biopolymers/www/request_form.html~~ Sequencing Request ~~Form~~]	+	#Fill out the [http://dnalims.mit.edu Sequencing Request WebForm]
	#*Write your name and e-mail, and the name of your lab		#*Write your name and e-mail, and the name of your lab
	#*Give the Template ID as whatever you want (probably the part number)		#*Give the Template ID as whatever you want (probably the part number)
	#*Primer ID is usually VF2 or VR1 (or an intermediate primer)		#*Primer ID is usually VF2 or VR1 (or an intermediate primer)
	#*Vector is whatever vector your part is on		#*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~~	+	#*Print 2 copies, bring one with your tubes to the biopolymers lab.
-	~~#*Keep one sheet for your records, and~~ bring one ~~sheet~~ 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).		#**For the Endy lab, remember to leave a copy in the Complete shelf of the lab filing cabinet (in 564d).
	#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).		#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)		#*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 (~~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.	+	#*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.
	#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.
	#*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.		#*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.