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Old 04-04-2013, 10:56 AM   #1
lmc
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Location: USA

Join Date: Jun 2010
Posts: 6
Default Necessary oligo modifications for home-made DNA-seq kit

Hi,
I am attempting to put together a set of adapters and blocking oligos for a multiplexed exome-capture experiment. I am following this: http://openwetware.org/images/1/10/Geller_exome.pdf
And a couple protocols from Roche/Bioos/other .

My questions are: 1) What modifications are necessary for the oligos that make the adapters? I have seen both with and without a penultimate phosphorothioate bond on one strand. Is the phosphorothioate bond necessary? What is it's purpose.
(There is also a 5' phos modification, I know this is necessary.)

2) For the blocking oligos, is there a preference in the 3' extension block? inverted-dT, dideoxy-C, or C3 spacer? I would like to use the C3 spacer since it is less expensive.

3) For both the blocking oligos & the adapters, what level/type of purification is required? HPLC or PAGE? I am assuming HPSF is in sufficient? How about "QuickLC"?

Thank you for your input!
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dna-seq, exome capture, exome-seq, library

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