I recently introduced a Lenti-Puro Guide vector containing a specific sgRNA sequence into a cell line. I would like to sequence the ddCas9-sgRNA cell lines to confirm that each sgRNA is accurate.
There should be sites flanking the MCS on the plasmid that containing standardized promoters (so sequencing cores don't have to try and track down a bunch of primers), T3/T7 phage promoters for run-off RNA polymerization, as well as multiple restriction sites. If you go to AddGene (or wherever you got it) and look at the plasmid sequence map you can use your discretion to determine which site you would like to use, assuming the lab that is sequencing it doesn't already have the standard sequencing primer set. If you're going to order custom oligos then remember that at the beginning of Sanger sequencing the resolution is not very good so you'll want to put some space between the forward primer and the region of interest.
Thank you Vincent!!!
There is a T7 promoter (3180 - 3199) and T3 (5993-6013), so I can design a forward primer specific to the T7 promoter or T3 promoter.
Is sequencing of a 10kb DNA region possible with the the location of the promoter relative to the gRNA scaffold region? I will be sequencing genomic DNA isolated from ddCas9-sgRNA AML12 cells.
e.g. by identifying primers detecting a sequense only found in the plasmid (e.g. mcs).
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