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Assay Design Cheat Sheet (DRAFT) by

AD Cheat Sheet for scarlett

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Transgenes (TG)

Basic Vector TG
Can target anywhere
TG with some homology
Target to least homologous region
Promot­er-­Vector TG
Target at promot­er-­vector junction
Mouse cDNA
Target at exon-exon junction (Be aware of intron size)
Human TG
Target to least homologous region

SNPs (MUT)

Basic SNP
Center SNPs in reporter oligos.
SNP based off AA change given
Locate AA change. Center SNPs in reporter oligos.
rs#
Locate rs#. Ensure reporter 1 is BL6 sequence. Center SNPs in reporter oligos.
Both reporters MUST be on the same strand or the assay will not work.

Humanized Models (KO)

Standard humanized KO model
Identify endogenous human junction and include 2-3 mismatches per oligo (WT and Mutant) if possible.
Humanized model with FRT or LoxP
Target flanking the FRT/LoxP which allows you to avoid homology
 

Knockouts (KO)

Standard KO Model
Target KO assay at 5' or 3' junction
Small CRISPR deletion
Balance KO probe across new junction
Large CRISPR deletion
Flank new junction
Designs for corres­ponding WT will be noted in the wildtype section.

KO landmarks

Start/Stop
Design WT at start or stop (depending on model). KO probe will be a generic.
Coding Region
Design anywhere in coding region. KO probe will be a generic
Restri­ction Site
Design according to restri­ction sites. KO probe will be a generic.
Coordi­nates
Design according to coordi­nates. KO probe will be a generic.
When in doubt - SEQUENCE.

Wildtypes (WT)

WT for small insertion (<4­00bp)
Balance probe (40/30) or do allele specific primer.
WT for large insertion (>4­00bp)
Can flank insertion site or have a probe across insertion site but do NOT need to balance.
WT where some bases were deleted (either via CRISPR or upon insertion of vector)
Target probe within deleted bases. Primers can be on either side.
 

Floxed (FL or MD)

Floxed allele with vector
Flank LoxP, use tearpin, or break the hairpin.
Floxed allele with minimal vector
Use tearpin or break the hairpin
Floxed allele when unsure which LoxP
Flank LoxP or do tearpin
Marker deleted floxed allele
Flank FRT/LoxP
Identify which LoxP you have. If you are breaking the hairpin you MUST know which LoxP to ensure you break in the correct direction.

Excised (EX)

EX with vector
Flank remaining LoxP or tearpin.
EX with minimal vector
Tearpin or can flank LoxP if large enough deletion created by Cre recomb­ina­tion.

CRISPR

NHEJ
Flank new junction created as long as it is >400bp
HDR
Must target 1 oligo outside of ssODN to be site specific.
Remember that CRISPR rules can apply to a variety of models including SNPs, Condit­ional lines, and even tradit­ional KOs. Please be aware if you are designing for CRISPR and utilize our best practices.