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Old 07-29-2010, 11:47 AM   #1
chainTerminator
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Default 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?
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Old 07-29-2010, 12:17 PM   #2
pmiguel
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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?

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Old 07-30-2010, 10:05 PM   #3
snetmcom
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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.
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Old 07-31-2010, 12:27 PM   #4
pmiguel
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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.

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