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Old 10-31-2009, 04:32 PM   #1
greigite
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Location: Cambridge, MA

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Default Elution volume after A-tailing?

The standard library prep protocol suggests to elute DNA with the MinElute kit following A-tailing and prior to adapter ligation, for a total volume of 10 ul. This 10 ul is then fed into the adapter ligation step. Is there any reason I couldn't elute the A-tailed product with a standard Qiaquick column (30 ul eluate) and just scale up reaction volumes in the ligation reaction? The post-ligation product is cleaned up on a Qiaquick column in any case. Seems simple enough to re-scale the adapter ligation reaction, but maybe I am missing something?
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Old 11-01-2009, 01:41 PM   #2
ScottC
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Eluting in a larger volume using standard Qiaquick column does indeed work in conjunction with a scaled-up ligation reaction..

I normally use minelute columns for this step, but once or twice when I've been in a hurry I've picked the wrong column and run my sample through before realising, only to realise the mistake at elution time. As you were planning to do, I just eluted in the larger volume (30uL) and scaled up the ligation... it worked just fine.

Out of interest... why are you looking at using the standard, rather than minelute columns? Just for the simplicity of using only one column type?

Cheers,

Scott.
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Old 11-01-2009, 01:45 PM   #3
greigite
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Quote:
Originally Posted by ScottC View Post
Eluting in a larger volume using standard Qiaquick column does indeed work in conjunction with a scaled-up ligation reaction..

I normally use minelute columns for this step, but once or twice when I've been in a hurry I've picked the wrong column and run my sample through before realising, only to realise the mistake at elution time. As you were planning to do, I just eluted in the larger volume (30uL) and scaled up the ligation... it worked just fine.

Out of interest... why are you looking at using the standard, rather than minelute columns? Just for the simplicity of using only one column type?

Cheers,

Scott.
Actually, it's just because someone used up all the minelute columns in the lab without replacing , and the stockroom is not open on weekends. No profound reason.
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