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Regulatory requirements vary by product. Please see product-specific literature to determine product use.

I would like to create a scrambled and/or point mutation negative control for my miR RNAi experiments. How should I do this? Product FAQ

Answer

In order to create a scramble miR RNAi negative control we recommend keeping 2-3 nt on each end of the guide RNA the same and scrambling the middle, then conducting BLAST to look for obvious problems. In order to create a point mutation miR RNA negative control, a single change may not be enough, but the best place to put it would be at nt 10 or 11 of the antisense sequence.

Answer Id:: E10002

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I'm seeing cytotoxic effects after transfection of my shRNA/miRNA construct. What is causing this? Product FAQ

Answer

You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.

Answer Id:: E10020

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I'm getting differently sized colonies after TOP10 E. coli transformation when using the miRNA lentiviral expression system. Which one should I use? Product FAQ

Answer

Some transformants may contain plasmids in which unwanted recombination has occurred between the 5' and 3' LTR. We recommend using our One Shot Stbl3 Chemically Competent E. coli cells, as they help in stabilizing lentiviral DNA containing direct repeats, and generally give rise to fewer unwanted recombinants. We recommend screening both colony sizes; however, in general, for lentiviral plasmids the small colonies tend to be the correct clones. Large colonies may have undergone a recombination event to delete part of the plasmid, thus allowing the cells to grow faster.

Answer Id:: E10026

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Can I freeze/thaw siRNA library stock plates? Product FAQ

Answer

Library stock plates are usually resuspended at less than or equal to 1 µM and stored at -20 degrees C or lower. Optimal storage is achieved at 10 µM stock concentrations. For Silencer siRNA, we recommend that the number of freeze/thaw cycles of the stock plates is limited to less than 10 for best results. For Silencer Select siRNA, we recommend that the number of freeze/thaw cycles of the stock plates is limited to less than 50 for best results.

Answer Id:: E10038

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Are the plates barcoded? Product FAQ

Answer

Yes, each plate of the library is provided with a unique barcode. No two plates have the same barcode identifier, and we can trace back samples on any plate given that barcode. The barcode identifiers are always listed in the electronic data file shipped with each library.
The barcode is located on the short end of the plate by column 12 (centered). This label includes the barcode, a human readable definition of that barcode and a plate name that helps users order plates within a library.

Answer Id:: E10056

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How should I have the siRNAs in my Invitrogen Custom siRNA Library plated? Product FAQ

Answer

For custom siRNA libraries, we can provide a number of plating options. Our most requested format is to have the last 1 or 2 columns empty of each 96-well plate, and to have different siRNAs to the same targets plated in separate plates in the same well location. The empty columns facilitate easy inclusion of any desired control in the final transfection plates. When different siRNAs to the same targets are plated in separate plates in the same well location, the siRNAs can easily be pooled with robotics or a multichannel pipettor. In addition, this type of format facilitates analysis by qRT-PCR.

Answer Id:: E10067

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What does CRISPR and CRISPR-Cas stand for? Product FAQ

Answer

CRISPR stands for clustered regularly interspaced short palindromic repeat; CRISPR-Cas (CRISPR-associated) systems are used for genome editing in various host organisms.

Answer Id:: E10116

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What is the difference between NHEJ- and HDR-mediated repair? Product FAQ

Answer

Both HDR (homology directed repair) and NHEJ (non-homologous end joining) are cellular mechanisms through which double-stranded DNA lesions are repaired. When a repair template is not present, NHEJ occurs to ligate double-stranded breaks, leaving behind insertion/deletion (indel) mutations. HDR is an alternative repair pathway in which a repair template is used to copy the sequence to the double-stranded break. You can, therefore, introduce specific nucleotide changes or DNA fragments into your target gene by using HDR with a repair template.

Answer Id:: E10122

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I am trying to target a big and multifunctional protein using CRISPR technology. To what site should I design my sgRNA? How can I check that the editing occurred? Product FAQ

Answer

The first few exons would be best (closer to the promoter, resulting in premature transcript termination). Since the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location, it is advisable to design and test a few target sites. Non-CRISPR-related mutations may be identified using gDNA isolated from non-CRISPR-treated cells as a control and performing a Invitrogen GeneArt Genomic Cleavage Detection Assay (https://www.thermofisher.com/order/catalog/product/A24372). Standard western blot analysis is a good measure for the verification of protein levels.

Answer Id:: E10128

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What happens to Cas9 after it performs its endonuclease activity? Product FAQ

Answer

Cas9 is transiently expressed and will therefore disappear over time with successive cell divisions.

Answer Id:: E10133

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When designing my primers for the CRISPR oligonucleotide, what level or purity is recommended? And do the oligonucleotides need to be phosphorylated? Product FAQ

Answer

5′-nonphosphorylated desalted oligonucleotides are fine. However, HPLC or higher purity will increase the cloning efficiency of your double-stranded oligonucleotide into the Invitrogen GeneArt CRISPR Nuclease vector.

Answer Id:: E10140

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How can I tell if my CRISPR genome editing experiment is working? Product FAQ

Answer

Cleavage efficiency can be detected using the Invitrogen GeneArt Genomic Cleavage Detection Assay. This assay relies on mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.

Answer Id:: E10154

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How efficient is the Cas9 mRNA format compared to the all-in-one CRISPR plasmid format? Product FAQ

Answer

In most cell types tested, this complete RNA format exhibits higher cleavage efficiency than the plasmid format. Additionally, the Cas9 mRNA format circumvents the need for cloning, has a smaller payload size, allows Cas9-to-gRNA dosage optimization, flexibility with multiplexing, and does not have any promoter constraints.

Answer Id:: E10160

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How does the GeneArt Genomic Cleavage Selection Kit work? Product FAQ

Answer

The vector in the kit has an OFP gene that is interrupted by the insertion of a cloning site for the target sequence of the programmable nuclease. The upstream sequence coding for the N-terminal portion of the OFP gene contains a region complementary to the 5′ end of the C-terminal region of the OFP gene. A stop codon after the N-terminal OFP sequence ensures no expression of the reporter prior to nuclease activity. The CD4 gene is out of frame for expression when the OFP gene is interrupted by the cloning site. Double-stranded breaks cause the complementary strands of the end sequences of the OFP gene to recombine, and OFP expression is restored. The CD4 gene is now in frame for expression and can be screened for via FACs analysis or Dynabeads magnetic bead selection.

Answer Id:: E10173

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What is the peak excitation and emission of OFP? What type of laser should I use? Product FAQ

Answer

OFP has peak excitation of 548 nm and emission of 560 nm. A 488 nm laser is recommended for efficient excitation. Standard 530/30, 574/26 and 603/48 emission filters are recommended for detection.

Answer Id:: E10179

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