Hello to all I have little doubt about ssodna as a repair template for my Knockin experiment. Actually, I've designed 147nt long ssodna but I do not figure it out. whether I have to order an only single strand donor template 5'-3' and transfect or I've to include reverse complementary 3'-5'as well and annealed both of them and make a Double Strand then transfect. I've read a couple of articles but still, I have confusion.
Could anyone provide the nice suggestion it would be appreciated?
Thank you
Hi Vinit, Thank you for writing to me.
But Origen is suggesting to prepare both sense and antisense ssodn separately and then annealed both of them. and transfect with CRISPR vector.
Here is a link to the protocol for gene tagging.
http://www.origene.com/assets/documents/cDNA/pCas-Guide_System_Validation.pdf
I agree with Vinit Shanbhag,
For design of the ssODNA you might also consider Richardson et al.'s Results-Part, discussing the HDR-Efficiency of DNA-Oligos with varying homology-lengths up-/downstream as well as target and nontarget strands of the dsBreak in comparison to a dsDonor.
Best regards,
Felix
Link to the paper: https://www.ncbi.nlm.nih.gov/pubmed/26789497
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