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With the CRISPR-Cas9 editing complex (DNA vector, mRNA or Protein), co-transfect a DNA repair template that contains high homology to the sequence of interest along with the desired sequence you would like to introduce into the DNA. By doing so HDR can occur, and your specific edits (mutation, insertion, etc.) can be incorporated into the genome.

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For transfection of standard non-modified siRNA, Silencer siRNA, Stealth RNAi siRNA, Silencer Select siRNA, Invitrogen Pre-miR Precursors, mirVana miRNA Mimics, Invitrogen Anti-miR Inhibitors, and mirVana miRNA Inhibitors, use only the Lipofectamine 3000 Reagent for efficient delivery to the cytoplasm. There is no need to use the P3000 Reagent.

For transfection of plasmid DNA, vector-based BLOCK-iT shRNA or miRNA, ViraPower HiPerform lentiviral expression vectors, or GeneArt CRISPR nuclease vectors, use Lipofectamine 3000 reagent with the P3000 Reagent for efficient delivery to the nucleus.

Please refer to the manual (https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_protocol.pdf) and to this scaling table (https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_scaling.pdf) for guidelines on amounts to use to prepare the transfection mixes.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

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The only difference between TOP10 and TOP10F' cells is that the latter contain the F' episome that carries the tetracycline resistance gene and allows isolation of single-stranded DNA from vectors that have an f1 origin of replication. The F' episome also carries the lacIq repressor for inducible expression from trc, tac, and lac promoters using IPTG. TOP10F' cells require IPTG induction for blue/white screening.

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Several ligation controls may be necessary to identify the source of ligation problems.

Recommendations for problems with no colonies after transformation:
1. Test the transformation efficiency of the competent cells using a supercoiled vector, or the control DNA provided with Invitrogen competent cells. Perform a transformation reaction and plate the number of cells that is expected to generate 50-100 colonies per plate, based on the anticipated transformation efficiency of the competent cells. The expected number of colonies should be seen, indicating that the competent cells are transforming with high efficiency. The control DNA provided with Invitrogen competent cells is supercoiled monomer; vector DNA preparations that contain other forms will not transform as efficiently. Transformation efficiencies will be up to 10-fold lower for ligated vectors than for intact control DNA.
2. Restriction endonuclease-digested, re-ligated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Re-ligation of vectors with cohesive ends should result in less than or equal to 50% of the number of colonies obtained with supercoiled vector DNA, indicating that the components of the ligation reaction are working; re-ligation of vectors with blunt ends should yield 10% to 20% of the number of colonies obtained with supercoiled vector DNA. This is an appropriate control only with vectors that have been digested with a single restriction endonuclease; double-digested vectors may not re-ligate because the ends are incompatible and the small DNA fragment that is released from between the two sites is sometimes lost during ethanol precipitation of the DNA.

For observation of a high number of background colonies:
1. Restriction endonuclease-digested vector. Perform a transformation with an amount of restriction enzyme-digested vector DNA equivalent to that contained in the fraction of the ligation reaction being used for the experimental transformations. Few or no colonies should be seen, indicating complete restriction endonuclease digestion of the vector. The presence of colonies indicates incomplete digestion of the vector that will cause a background of colonies containing non-recombinant vector in the experimental transformations.
2. Restriction endonuclease-digested, dephosphorylated, re-ligated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Few or no colonies should be observed, indicating complete dephosphorylation of the vector - a dephosphorylated vector should not be re-ligated by T4 DNA ligase.
3. No DNA transformation control. Perform a mock transformation of competent cells, to which no DNA is added. No colonies should be seen, indicating that the selection antibiotic on the agar plates is potent and that the competent cells are pure.

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We do not offer any of the MultiSite Gateway vectors as standalone products. They are only available as part of the kits.

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Yes, we carry pcDNA6.2/V5-PL-DEST, that is promoter-less. You would use this vector if you wanted to clone in your own 5' promoter using the MultiSite Gateway Pro kit.

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– Check whether the correct Clonase enzyme was used and whether it was functional (make sure that LR Clonase II Plus was used).
– Check whether the correct combination of Entry vectors and Destination vector was used.
– Ensure that the correct Entry clones were used (and that they were sequenced).
– Check if the recommended amount of Entry vector and Destination vector was used in the reaction.
– Perform the LR reaction positive controls to determine which Entry clone may be faulty (note that MultiSite GW 3-Fragment kit only comes with a single control vector, pMS/GW, that is used to make all control pENTR vectors).
– Check whether the correct antibiotic was used for selection.
– Check whether the att site sequences in the Destination vector are correct.
– Increase the incubation time up to 18 hours.

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We carry the pVAX1 vector, Cat. No. V26020, engineered according to FDA guidelines, for this purpose. In this vector, eukaryotic DNA sequences are limited to those required for expression in order to minimize the possibility of chromosomal integration. It has the kanamycin resistance marker for selection in E. coli instead of ampicillin, which has been reported to cause an allergic response in some individuals.

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To test for the presence of ligation inhibitors, perform a ligation reaction in which some of the vector or insert DNA is included along with some marker DNA such as lambda DNA/Hind III Fragments. If ligation of the DNA marker fragments occurs alone but is not observed when other DNA is added, then a diffusible inhibitor is present in the vector or insert DNA.

To purify and remove inhibitors, extract the DNA with buffer-saturated phenol, then extract with chloroform:isoamyl alcohol, and precipitate with ammonium acetate and ethanol. Be sure that the DNA is free of phenol and that the phosphate concentration is less than 25 mM and the NaCl concentration is less than 50 mM. Also, be sure that the DNA is free of contaminating DNA that might compete for ligation to the insert or vector (e.g., linker fragments, DNA fragments from which the insert was completely purified).

If restriction endonucleases are present, causing redigestion of ligated products, your ligation will also be inhibited. After digestion of the vector and insert DNA, remove restriction endonucleases by extraction with buffer-saturated phenol, extraction with chloroform:isoamyl alcohol, and ethanol precipitation.

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pDONR 221 is provided as a positive control for the BP recombination reaction, but cannot be used to generate multi-fragment entry clones in the MultiSite Gateway Pro kit.

For the LR reaction, all Entry clones can be substituted with the supplied Control Entry clones. The number of colonies expected and/or the phenotype of the resulting clones are used to determine the efficiency of the LR recombination reaction. In the unlikely event that the attR sites in the destination vector are incorrect, then the LR reaction will result in zero clones. By performing one-by-one vector substitution reactions, the entry clone that is flawed can be identified.
– For the 2-Fragment LR Clonase® II Plus Positive Control recombination reaction, 2,000 - 15,000 colonies are expected if the entire 10 µL LR reaction is transformed.
– For the 3-Fragment LR Clonase® II Plus Positive Control recombination reaction, 1,000 - 5,000 colonies are expected if the entire 10 µL LR reaction is transformed. – For the 4-Fragment LR Clonase® II Plus Positive Control recombination reaction, 50 - 500 colonies are expected if the entire 10 µL LR reaction is transformed.

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The MultiSite Gateway Pro 2.0, 3.0, and 4.0 kits have been discontinued as of December 31, 2015. The alternative product is the MultiSite Gateway Pro Plus Kit, Cat. No. 12537100, for cloning of up to 4 DNA fragments.

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