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Can I purchase the 5X LR Clonase buffer or 5X BP Clonase buffer separately? Product FAQ

Answer

We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.

Answer Id:: E9855

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For how long are the reagents in the Invitrogen GeneArt CRISPR Nuclease Vector kit guaranteed? Product FAQ

Answer

All reagents are guaranteed to be stable for 6 months when properly stored. The vector, buffers, control oligo, plasmid, water, and sequencing primers should be stored at -20°C.

Answer Id:: E10151

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What is the buffer composition for the Platinum Cas9 protein? Product FAQ

Answer

The buffer composition for the Platinum Cas9 protein is as follows:
10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.6 mM TCEP, 50% glycerol. Please note that we can make custom formulations if necessary.

Answer Id:: E11533

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What is the protocol for Western transfer after native electrophoresis? Is it possible to transfer a native protein that has a higher pI than the pH of the transfer buffer? Product FAQ

Answer

Western transfer of native proteins is difficult due to the relatively low charge density of most native protein molecules. Therefore, the conditions most suitable for transfer of individual proteins vary. In a neutral-pH transfer buffer, proteins with low pI are expected to transfer toward the anode, as in denaturing electrophoresis, while proteins with high pI are expected to transfer toward the cathode.

First, consider whether native transfer is really required. Just because the gel electrophoresis was run under native conditions does not mean that the transfer needs to be native as well. Following a native gel run, proteins will be separated based on their native mobilities in the gel. Transferring with an SDS-containing buffer will not affect their positions on the gel - it will simply denature the proteins and make transfer much more efficient in most cases.

Whether you choose to transfer proteins under native conditions or under SDS (denaturing) conditions depends on the planned downstream analysis and whether your detection method requires proteins to be in their native state. If you do decide that you should transfer your proteins in native mode, and you are using Tris-Glycine gels, here are a few things to try:

1) Use the normal transfer buffer and place membranes on BOTH sides of the gel. Any proteins that are more basic than the pH of the transfer buffer will be captured on the membrane placed on the cathodic side. Both membranes can then be developed in the same manner.

2) Increase the pH of the transfer buffer to 9.2, which will allow all proteins with pI below 9.2 to transfer toward the anode.

3) Prior to blotting, incubate the gel for 15 min in Tris-Glycine Transfer Buffer with 0.1% SDS. During the subsequent transfer, use transfer buffer without SDS. The small amount of SDS in the initial incubation may give the proteins enough charge to move unidirectionally towards the anode and, in most cases, should not denature the proteins.

Blue Native gel electrophoresis is another method that can simplify the blotting of native proteins. G-250 dye is added to the samples, which imparts a slight negative charge to the proteins without denaturing them. Blotting after Blue Native electrophoresis will only require one PVDF membrane on the anode side, NuPAGE Transfer Buffer, and no SDS. For more details on the Invitrogen Blue Native gel technology, see NativePAGE Invitrogen Bis-Tris Gels in our online catalog.

Answer Id:: E3271

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What is the storage buffer composition for TrueCut Cas9 Protein v2? Product FAQ

Answer

The storage buffer composition for TrueCut Cas9 Protein v2 is as follows:
15 mM Tris-HCl pH 8.0, 150 mM NaCl, 1.0 mM TCEP, 50% glycerol.

Answer Id:: E16346

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What is the molecular weight of the L-arabinose included in the pBAD kits? Product FAQ

Answer

The molecular weight of arabinose is 150.1 g/mol.

Answer Id:: E3249

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What are the best conditions for double digests with conventional Thermo Scientific restriction endonucleases? Product FAQ

Answer

First, please note that we changed our restriction enzymes and buffer formulations in 2010. The REact buffers 1-10 were discontinued in favor of a smaller group of universal buffers: B, G, O, R and Tango (Y). The new buffers are not compatible with older restriction enzymes, and it is not recommended to do a double digest with an old enzyme (with REact buffer) and a new enzyme.

When performing any double digest, there may be buffer incompatibilities and enzyme steric hindrance problems. These can be avoided by performing sequential digests, separated by buffer exchange or chloroform extraction and ethanol precipitation. However, if these issues are understood and a double digest will be performed, you can evaluate enzyme combinations using our buffer compatibility chart: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/conventional-restriction-enzymes-thermo-scientific/reaction-conditions-for-restriction-enzymes.html.

Answer Id:: E2929

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Can I use a mini-prep kit for propagation of my lentiviral construct? Product FAQ

Answer

We do not recommend using a mini-prep kit for propagation of lentiviral constructs because the DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004), both of which contain 10 mM EDTA in the Resuspension Buffer. Since lentiviral DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield-basically, grow cells slowly, use fewer cells per column, and use 100 mL lentivirus culture for each DNA midi-prep.

Note: If you are going to be mini-prepping the lentiviral plasmid during the cloning/colony screening processes, we recommend using the PureLink HQ Mini Kit (Cat. No. K210001) and following the manual protocol with one change: only a single elution with 50 mL TE, pH 8.0 buffer. The typical yield with this method is normally pretty low: 100-150 ng/mL (i.e., 5-7 mg total). The OD 260/280 is typically between 1.8 and 2.1.

Answer Id:: E9293

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Are there any gene editing and gene optimization functions available when ordering GeneArt Strings DNA fragments? Product FAQ

Answer

Yes. We recommend using the GeneArt web portal for ordering, where you can use the free gene editing and optimization functions to adjust the sequence to meet your needs. After editing and optimization, please check again that the final sequences of the fragments are exactly as needed for further processing in your lab (e.g., potential homologous overlaps of the fragments or restriction sites and buffer bases needed for cloning).

Answer Id:: E7655

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When I receive my CRISPR oligonucleotide, what should I resuspend it in? Product FAQ

Answer

Either nuclease-free water or TE buffer is fine, but please anneal using the appropriate Oligonucleotide Annealing Buffer for your annealing reaction. Subsequently, please serially dilute your primers in a final concentration of 1X Oligonucleotide Annealing Buffer.

Answer Id:: E10141

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I'm getting low to no colonies after transformation. Why? Product FAQ

Answer

Some possible causes and remedies are:
- Ligase function is poor. Check the age of the ligase and function of the buffer.
- Competent cells are not transforming. Test the efficiency of the cells with a control supercoiled vector, such as puc19.
- Both molecules were de-phosphorylated.
- Inhibition of ligation by restriction enzymes and residual buffer. Try transformation of uncut vector, clean up restriction with phenol, or carry out PCR cleanup/gel extraction before ligation.
- Incorrect antibiotic selection used. Check the plasmid and plates and make sure concentration of antibiotic used is correct.

If nothing above applies, low to no colonies may be due to instability of the insert DNA in your competent cells. In this case, E. coli strains such as Stbl2, Stbl3, or Stbl4 have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.

Answer Id:: E6667

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I used pDisplay vector to express a cell surface receptor protein. Should I be concerned about digestion of the protein during trypsinization of the cells? Product FAQ

Answer

Trypsin digests proteins at arginine or lysine residues, and it is widely accepted that the extracellular domains of membrane proteins are digested upon trypsinization of cells. But typically these proteins are rapidly replaced once the trypsin is removed. As an alternative to trypsin, you can remove most adherent cells using Versene (Cat. No. 15040066), which is a sterile 0.5 mM EDTA solution in PBS, and/or scraping. The EDTA chelates any free Mg2+ or Ca2+ ions, which are necessary for maintaining many cell attachments. Cell Dissociation Buffer, Enzyme-free, Hank's-based (Cat. No. 13150016) or PBS-based (Cat. No. 13151014), which are cocktails of chelating agents, are also fine for this application and may be more effective than EDTA alone, and would be unlikely to adversely affect the receptor protein.

Answer Id:: E9208

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The tube containing the TOPO TA vector arrived yellow instead of the usual red color. What does this indicate? Will this color change affect the activity of the Topoisomerase? Product FAQ

Answer

Phenol red is added to the TOPO vector (pCRII, pCR2.1, and pCR4), mostly to make it more visible in the tube. The color of the solution is generally pink at room temperature, but at a low pH phenol red will turn yellow. We have tested acidic conditions (low pH) for the TOPO ligation reaction and found that the activity is not affected. At a pH 2.0, there was no observed decrease in efficiency. However, if the TOPO vector turns dark red, or is blue after adding the PCR product, then the PCR product buffer is too basic (pH 9). High pH does have a negative effect on the TOPO vectors, and the number of resulting colonies in this case will drop.

Note: The Directional TOPO and Zero Blunt TOPO vectors contain bromophenol blue dye rather than phenol red, which results in a blue-colored solution. Consult the product manual to confirm which dye is added to your vector, or contact Technical Support for more information.

Answer Id:: E3341

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What is the composition of the ViraPower lentiviral packaging mix? Product FAQ

Answer

The ViraPower lentiviral packaging mix is an optimized mix of three packaging plasmids: pLP1, pLP2 and pLP/VSVG, supplied in TE buffer, pH 8.0, at a concentration of 1 µg/µL. These plasmids supply the helper functions as well as structural and replication proteins in trans that are required to produce lentivirus using our ViraPower lentiviral expression vectors.

Answer Id:: E6398

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I received my TOPO vector and the solution is colored. Is it okay to use? Product FAQ

Answer

TOPO and TOPO TA vectors (non-directional) have phenol red dye added. The color should be pink (or yellow) at room temperature. If it turns blue when PCR product is added, the PCR product buffer is too basic and the number of transformed colonies will drop. When the solution is yellow, it signifies an acidic pH. At a pH 2.0, TOPO vectors still maintain high cloning efficiency. Directional TOPO and Zero Blunt TOPO vectors have bromophenol blue dye added.

Answer Id:: E6735

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