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View the relevant questions below:

TALs

We have observed that as little as 3 bp mismatch affects the binding of the TALs. We recommend that there be an exact match between the design and the binding site. If you would like some help with the design, please email geneartsupport@lifetech.com

The 19 bp binding domains perform better for the nucleases. The binding sites do not need to be the same size; however, best performance for the nucleases is with the 19 bp binding domains.

While our Invitrogen™ GeneArt™ Precision TALs required a T at the 5’ end and 13–18 bp spacing between the forward and reverse TAL effectors for proper pairing of Fok1 nucleases, the Invitrogen™ GeneArt™ PerfectMatch TALs allow for targeting of any sequence across the genome and eliminates the 5’ T constraints. Additionally, the spacing between the two effectors is optimal at 15–16 bp.

We do offer a multiple cloning site sequence in the place of the effector domain sequence for our TAL MCS entry vector. This option allows you to insert any protein-coding sequence, and allows your resulting TAL protein to deliver the effector in a sequence-specific manner anywhere in the genome. We also provide gene synthesis services to generate any effector domain for which you don’t have a template.

Invitrogen™ GeneArt™ Genomic Cleavage Selection Kit

Please see the possible reasons and suggestions below:

Reason

Solution

Single-stranded (ss) oligonucleotide designed incorrectly

Make sure that each ss oligonucleotide contains the 4 nucleotides on the 5’ end required for cloning into the GeneArt Cleavage Selection vector:

Top strand includes AATT on the 5’ end

Bottom strand includes CTAG on the 5’ end

Double-stranded (ds) oligonucleotides were degraded

Store the 50 nM ds oligonucleotide stock in 1X ligation buffer at –20°C

Avoid repeat freeze/thaw cycles, aliquot 50 nM ds oligonucleotides stock and store at –20°C

Oligonucleotide annealing reaction was inefficient

Ensure that the annealing reaction was performed as directed

If ambient temperature is >25°C, incubate the annealing reaction in a 25°C incubator

This can occur if:

  • Oligos are designed incorrectly; make sure to include specific sequences for TALEN/CRISPR target
  • Transfection efficiency is too low; optimize conditions or use Invitrogen™ Lipofectamine™ 3000 or 2000 reagent for best results
  • Cell line incompatible; OFP does not express well in certain cell lines; we recommend using 293FT cells as a test for cleavage activity

Too much OFP background can occur if there is plasmid contamination, and/or can be cell line– and target-dependent. Make sure to pick single clones when culturing the cleavage selection plasmid, or try to reduce the amount of vector that is included in the transfection.

Results are locus dependent. We recommend using the Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit (Cat. No. A24372) to verify cleavage on the endogenous genomic locus.

Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit

Please see the table below for common problems, possible causes, and recommendations:

DNA band appearance

Possible cause

Recommendation

Smear

Lysate is too concentrated

Dilute lysate 2-fold to 4-fold and repeat the PCR reaction.

Too faint

Lysate is too dilute

Double the amount of lysate in the PCR reaction. Do not use more than 4 µL of lysate in the PCR reaction. Lysate can inhibit the PCR reaction.

Disparity in band intensity between amplicons

Lysate concentrations vary between samples

Purify the PCR products with Invitrogen™ PureLink™ PCR Purification Kit. For best results in comparing samples, purify PCR and use the same quantity of DNA in each cleavage assay; 50 ng to 100 ng of DNA is enough for each reaction.

No PCR product

Poor PCR primer design or GC-rich region

Redesign primers that are 18–22 bp, have 45–60% GC content, and a 52–58°C Tm range. For GC-rich regions, add 1–10 µL of 360 GC Enhancer in a 50 µL reaction and repeat the PCR amplification.

Please see the table below for common problems, possible causes, and recommendations:

Problem

 Possible cause

Recommendation

No cleavage band visible

Nucleases unable to access target sequence or unable to cleave at target site.

Transfection efficiency too low.

Design new targeting strategy at nearby sequences.

Genomic modification too low.

Optimize transfection protocol.

Omitted denaturing and reannealing step.

Use Kit Control Template and Primers to verify the kit components and protocol

Difficulty in analyzing gel data

Background interferes with the measurements of cleaved bands.

Redesign the PCR primers to product a distinct cleaved binding pattern.

Nonspecific cleavage bands

Intricate mutations at the target site.

Digestion incubation too long.

Too much Detection Enzyme added.

Nonspecific cleavage by Detection Enzyme for certain target loci.

Redesign PCR primers to amplify target sequence. 

Use lysate from mock transfected cells or cells transfected with irrelevant plasmids as negative control to distinguish background from specific cleavage.