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Old 05-02-2015, 02:38 AM   #1
blackknight
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Location: Gothenburg, Sweden

Join Date: Feb 2014
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Default GUIDE-seq oligo design

Hi everyone,

I am trying to understand how the primers were designed for the GUIDE-seq protocol:

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
http://www.nature.com/nbt/journal/v3.../nbt.3117.html

The protocol states that I should use either sense or antisense primers, but it looks like to me that if I only use the sense primers I will get a very small PCR product only containing the ODN and other parts of the oligo and no information about the sequence next to the ODN.

To me it looks like that GSP1-sense does not fit with the GSP2-sense primer. The GSP1-sense primer aligns with the GSP2-anti oligo.

Can anyone explain how it works? I have attached a PDF of my alignments.

Thanks,

Anders
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File Type: pdf ODN PCR.pdf (156.1 KB, 32 views)
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Old 06-01-2018, 01:15 PM   #2
captainentropy
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Location: San Francisco Bay Area

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Default

You'll capture DNA adjacent to one-side of the oligo integration site whichever direction you choose to use. We went with the negative strand primer as GSP1(-) will have a longer contact with the ODN (29 bases vs. 22). Our thinking was the longer stretch will be marginally more stable and thus improve the capture. But we got good data with the (+) as well.

I need to write things out to see what's going on with these types of assays so here's what I was constructed. I think it's accurate, so if you find an error just PM me.
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File Type: pdf GUIDE-seq_scheme.pdf (18.9 KB, 6 views)
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