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Old 01-10-2018, 02:37 AM   #1
liaoyunshi
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Location: Hong Kong

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Default how to self order illumina library and reagent

I am a new for Illumina, recently our lab want to prepare the illumina library by ourselves, without using the current kit. So I have the following questions would like to ask for your help.

1. Is it feasible to directly order the Illumina adapters from the oligos synthesis company? If yes, the adapters should be in which purified level? HPLC?

2. Illumina need to do PCR to amplify the fragmented library. For the primer, which purified level should be set? Also HPLC?

Thanks.
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Old 01-10-2018, 08:29 AM   #2
pmiguel
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Yes, this is possible. Some companies will even anneal the adapters for you and ship them in that format.
Usually you would want to put a phosphorothioate linkage for the 3' T overhang base to reduce it's susceptibility to some exonucleases.

(Actually, there is verbiage in one of Illumina's patents about actually treating their adapters with an exonuclease because one of the phosphorothioate linkage stereoisomers was resistant to cleavage but the other was not. So they would digest the adapter and then do PAGE purification to remove the susceptible linkage adapters. Do they still do that?)

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