Big Dye Terminator

chainTerminator · 07-29-2010, 11:47 AM

Hello All-

Does anybody know how to make their Ready reaction mix and 5x Sequencing buffer? We've been trying to reverse engineer it but no luck. We use ABI's v3.1. Its pretty costly so we're trying to make our own! Any ideas?

pmiguel · 07-29-2010, 12:17 PM

Trying to resist answering first gen sequence question...
Sorry, ECO, it is not possible for me. Maybe you could create a first gen forum?

Okay, this is a "next gen" sequencing forum, not a "this gen" forum. That said, I do not think anyone makes the actual big dye "ready reaction" mix in their lab.

The so-called "5x" buffer is another story, but not one with a happy ending. First, I have no idea why it is called "5x" buffer, when one uses it at 2.5x. But that has always been its name. So, here is the story: prior to the ".1" versions of Big Dye, 5x buffer was just Tris and MgCl2. You could make it yourself and we did. With the introduction of BD 1.1 and 3.1, however, something changed with the new formulation and the old 5x buffer no longer worked. (5% v/v DMSO also became detrimental, rather than helpful in the sequencing reactions.)

What was the change? I don't know. (That is the sad ending. Sorry.) Some sort of non-ionic detergent maybe?

--
Phillip

snetmcom · 07-30-2010, 10:05 PM

yeah. the ABRF discussion group will likely be more help.

and there is a public formulation of the buffer, though you really shouldn't be going through it faster than the big dye.

pmiguel · 07-31-2010, 12:27 PM

Hi snetmcom,
Where is the public formulation? Are you sure it isn't the v .0 5x recipe? That has been long known. The v .1 5x recipe is another matter. At this juncture I don't see ever needing to make it -- we have plenty. But depending on the BD dilution one uses, the 5x buffer included with the Big Dye will not necessarily be enough.

But I would like to know the recipe for 1.1 and 3.1 5x buffer -- just to satisfy my curiosity.

--
Phillip

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07-29-2010, 11:47 AM	#1
chainTerminator Junior Member Location: Cambridge, MA Join Date: Jul 2010 Posts: 2	Big Dye Terminator Hello All- Does anybody know how to make their Ready reaction mix and 5x Sequencing buffer? We've been trying to reverse engineer it but no luck. We use ABI's v3.1. Its pretty costly so we're trying to make our own! Any ideas?

07-29-2010, 12:17 PM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Trying to resist answering first gen sequence question... Sorry, ECO, it is not possible for me. Maybe you could create a first gen forum? Okay, this is a "next gen" sequencing forum, not a "this gen" forum. That said, I do not think anyone makes the actual big dye "ready reaction" mix in their lab. The so-called "5x" buffer is another story, but not one with a happy ending. First, I have no idea why it is called "5x" buffer, when one uses it at 2.5x. But that has always been its name. So, here is the story: prior to the ".1" versions of Big Dye, 5x buffer was just Tris and MgCl2. You could make it yourself and we did. With the introduction of BD 1.1 and 3.1, however, something changed with the new formulation and the old 5x buffer no longer worked. (5% v/v DMSO also became detrimental, rather than helpful in the sequencing reactions.) What was the change? I don't know. (That is the sad ending. Sorry.) Some sort of non-ionic detergent maybe? -- Phillip

07-30-2010, 10:05 PM	#3
snetmcom Senior Member Location: USA Join Date: Oct 2008 Posts: 158	yeah. the ABRF discussion group will likely be more help. and there is a public formulation of the buffer, though you really shouldn't be going through it faster than the big dye.

07-31-2010, 12:27 PM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Hi snetmcom, Where is the public formulation? Are you sure it isn't the v .0 5x recipe? That has been long known. The v .1 5x recipe is another matter. At this juncture I don't see ever needing to make it -- we have plenty. But depending on the BD dilution one uses, the 5x buffer included with the Big Dye will not necessarily be enough. But I would like to know the recipe for 1.1 and 3.1 5x buffer -- just to satisfy my curiosity. -- Phillip