designing primers for surveyor

In the Ann Ran et al., 2013, Nat Protocols paper, it said that the online gRNA design tools also provides optimized primers for surveyor assay. However, I could not find online tools for designing primers to do Surveyor.

Are there available tools online?


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Brown Garudaover 6 years ago

I am agree with you. I was also looking for same things. I think there is no tool to design the primers for SURVEYOR. But in his main paper they mentioned that 'SURVEYOR primers should be designed to amplify 200–400 bp on either side of the Cas9 target (for a total amplicon 400–800 bp long). I hope this will help.

If you did know any tool in future, please let me know.

Jade Vampireover 6 years ago

I usually copy around 300-400bp upstream and downstream from the targetsite from the genomic DNA-sequence (ENSEMBL) and design Primers with the "usual" tools--> like Primer3Plus...If the PCR works well (e.g. single decently strong band) I use them for sequencing the region for SNPs + they serve well as primers for the surveyor assay. Hope that helps...

Brown Garudaover 6 years ago

Thanks.

Sapphire Unicornover 6 years ago

CHOPCHOP will design both gRNAs and primers for you for Surveyor or T7E1 assays.

http://chopchop.cbu.uib.no/

Green Gremlinalmost 5 years ago

I am completely new to the field of CRISPR/Cas9 and I need to design some primers for the T7 endonuclease assay from IDT.

Could somebody guide me how to do that? Do you know any company that could help me doing that?

Jade Titanalmost 5 years ago

I'd suggest you use primer3 (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi, or any of the other versions). Set the amplicon size to 500-1000 bp. then design primers around your region of interest such that the sgRNA is ~1/3 to 1/4 the distance from either end. the reason you do this is that after you add the T7 or Surveyor, it will cleave mismatches (where the sgRNA is making DSBs and indels are forming). So when you run it on a high res gel (TBE SDS PAGE is what i usually use, 4-12% or 10%), then you want to be able to see the "split" products. if you made the primers so that the sgrna was in the middle, you'd only see one band (because they'd be the same size), so putting the primers so that the sgRNA is located at a position such that cleaved it will make two bands (1/3---2/3 the total) lets you see clearly two bands when running on a gel. Does that make sense?

Green Gremlinover 4 years ago

I would like to know where I can find detailed information about primers design. I have desinged on my own the primers and then I also checked which Surveyor primers are recommended in the CHOPCHOP for detecting missmatches and I found my primers there.

I performed a PCR gradient for the annealing temperature. After validating my primers on non transfected cell lines and scrambled control transfected cell lines, I chose the proper Tannealing and followed the instructions of the At-R Genome Editing Detection kit from IDT. I am also using the HotStart DNA Polymerase from Roche as they recommend. In all the cases I am isolating the DNA by using the QUICK Extract DNA solution mix from Epicenter.

It is weird because the controls from the kit give me a band of around 10,000 bp and I am unable to observe the 3 bands as expected in control B. Moreover none of my cells showed insertions or deletions (I only see the same band in all).

I was wondering if the mistake could be due to the design of the primers (which I do not think so but i am not completely confident they are right because I am new on it) or if somebody could just help me out to solve this issue.

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