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饿的神啊

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[求助] hairpin for VIGS 已有1人参与

1、像这种反向互补序列是直接送公司合成的，还是设计一半的引物，然后让引物退火成双链？
2、如果是直接合成的，插入载体种还要同时做正、反向插入吗？

以下是原文描述，我不是很懂，求解。
The oligonucleotides PDS-54s and PDS-54as, corresponding to nt 1176–1220 of the Arabidopsis PDS cds, were hybridized to each other and cloned into the SnaBI site of E17-S in the sense or antisense orientation to generate E17-PDS-s and E17-PDS- as, respectively. The oligonucleotides PDS-IR37 and PDS-IR41, corresponding to nt 1176–1212 of the Arabidopsis PDS cds, were hybridized to each other and cloned into the SnaBI site of E17-S
to generate E17-PDS-IR.

The other VIGS vector constructs (pTY-PDS48-IR, pTY-PDS52-IR, pTY-PDS56-IR, pTY-PDS60-IR, pTY-PDS64-IR, pTY-PDS70-IR, pTYPDS76-IR, pTY-PDS2-IR and pTY-LFY-IR) were obtained by cloning the self-hybridized palindromic oligonucleotides PDS48, PDS52, PDS56, PDS60, PDS64, PDS70, PDS76, PDS2-IR and LFY-IR (corresponding to nt 911–949 of the LFY cds), respectively, into the SnaBI site of pTY-S.

This small size therefore allows the introduction of foreign sequences in the viral genome by direct cloning of doublestranded synthetic oligonucleotides. To target a specific gene, we therefore advise that a self-hybridizing palindromic oligonucleotide of 76–80 nt, identical to the target gene
sequence, be inserted into the SnaB1 site of pTY-S, so that hairpin RNAs bearing a stem of 36–38 nt and a 4 nt loop could be generated.

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1楼 2017-10-10 12:35:13

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yudaoqian88

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5楼: Originally posted by 饿的神啊 at 2017-10-11 09:30:04
比如说图中第一条PDS48，它其实左边是一条24bp的寡核苷酸，右边是反向互补序列，那么合成的时候只合成24bp？那退火成双链是引物二聚体吧，也形成不了双链啊？还是说直接合成48bp的寡核苷酸？...

双链倒来倒去很乱，容易迷糊

直接合成全长就可以了