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Old 06-09-2014, 11:23 AM   #29
jwfoley
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Location: Stanford

Join Date: Jun 2009
Posts: 179
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Quote:
the cost/sample of the "batch of expensive oligos" is negligible...you donīt have to order buckets of it...
Oh, maybe that's why we see this differently. With my supplier, the smallest possible order of the TSO is about 50 micrograms, for about USD 500. Obviously that's enough for hundreds or thousands of libraries, but if you only want to make a dozen, it's a pretty big commitment, especially when the rest of the reagents are off-the-shelf and/or can be bought in much smaller effective amounts. So doubling the amount of TSO you must purchase, just to try a risky way of getting a slight yield increase that may require falling back to the foolproof way anyhow, is rather unattractive if you only plan to do one experiment rather than start a service center. Otherwise you might just buy the kit.

Quote:
at least until you prove the rest of the world wrong
I don't know what the rest of the world does, but the STRT-seq folks aren't even outside your institution: "Critical: This oligo must be 5′ biotinylated to prevent template switching at its 5′ end (which would generate useless background reads)." (doi:10.1038/nprot.2012.022) At any rate, your own protocol's note that unblocked oligos sometimes result in concatamers is more than enough to persuade me not to waste any time on them.
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