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

Product FAQ

Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Answer

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

Answer Id: E3098

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How can unstable or toxic DNA inserts be maintained in bacteria?

Answer

There are a few steps you can take to improve stability of clones with difficult-to-maintain inserts. Supplement the medium with extra nutrients (e.g., add 20-30 mM glucose to Terrific Broth) or try a vector that has a reduced copy number (e.g., pBR322). Some clones can exhibit a high degree of deletions; this is usually a result of the clones having long terminal repeat (LTR) sequences or regions with high secondary structure. To overcome this problem, the cells can be grown at 30°C or ambient temperature (in LB or in a nutrient rich broth like Terrific Broth). Do not to let the cells reach late stationary phase in liquid culture. Alternatively, transform into cells that maintain unstable sequences such as Stbl2, Stbl3, or Stbl4 cells.

Answer Id: E3099

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How stable is the ULYSIS labeled DNA to high temperature?

Answer

An oligonucleotide labeled with a ULYSIS Nucleic Acid Labeling Kit should survive 100°C for 5 minutes, and storage at 68°C overnight should also not cause any dissociation of the complex.

Answer Id: E8093

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Can AgPath-ID One-Step RT-PCR Reagents (Cat. No. 4387424) be used with fast cycling conditions?

Answer

Generally, we recommend using this master mix with standard cycling conditions, but depending on the assay being used, you could use fast cycling conditions with optimization.

Answer Id: E16418

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

Answer

Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

Answer Id: E3100

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I need to purify a cloned plasmid, which I will be transfecting into a cell line for protein expression. I have both silica and anion exchange columns available. Which would you recommend as the better option for my application?

Answer

Anion exchange purification is recommended for higher purity and lower endotoxin levels. Silica-based purification is not optimal for transfection, as there is a higher level of endotoxins and impurities. Larger plasmids also work better with anion exchange columns.

Answer Id: E7738

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Can the ULYSIS kits be used on probes longer than 1,000 base pairs or even plasmids?

Answer

It might be possible to label larger probes with the ULYSIS Nucleic Acid Labeling Kits, but the dye will likely need to be diluted to avoid (or at least reduce) problems with aggregation. Refer to this publication: Coelho-Castelo AA, Santos Junior RR, Bonato VL et al. (2003) B-lymphocytes in bone marrow or lymph nodes can take up plasmid DNA after intramuscular delivery. Hum Gene Ther 14(13):1279-1285.

Answer Id: E8094

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Where can I find information about how to analyze data generated using the VetMAX-Plus One-Step RT-PCR kit (Cat. No. 4415328)?

Answer

Please see page 6 of this protocol (https://tools.thermofisher.com/content/sfs/manuals/cms_063757.pdf) for details.

Answer Id: E16419

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Does the method of generating lipid-DNA complex affect transfection efficiency?

Answer

YES. Please follow the recommended procedure for each one of the reagents, found in the product manual.

Answer Id: E3127

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Can encapsulated phagemid DNA or M13 phage be used to infect bacteria?

Answer

Single-stranded DNA viral particles like M13 require the presence of an F pilus in order to infect E. coli. This criterion is met by TOP10F', DH5? F'IQ, INV?F', Stbl4, OmniMAX2-T1 and DH12S cells. These cells are not traD mutants, which effectively allows the cells to retain the F' episome. Transforming single-stranded DNA can cause a 100- to 1,000-fold reduction in efficiency compared to viral particles.

Answer Id: E3101

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What are endotoxins and why are their levels important for transfection?

Answer

Endotoxins are typically any cell-associated bacterial toxins that are part of the outer surface of the cell wall of gram-negative bacteria. Endotoxins can influence cell growth, cell differentiation, contractility, and protein expression in mammalian cells. Endotoxins are released during bacterial lysis, and they can subsequently reduce transfection efficiency and protein expression levels. Please review the following article for more information about endotoxins: Butash KA et al. (2000) Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency. Biotechniques 29(3): 610-614, 616, 618-619.

Answer Id: E7739

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Do you have any tips on using the ULYSIS Nucleic Acid Labeling Kits for RNA labeling?

Answer

A preliminary protocol modifies our DNA-labeling protocol: Do not nuclease-treat the RNA, but label it directly by incubating for 10 minutes at 90°C or 15 minutes at 85°C. Add 2 µg of glycogen for every 1 µg of RNA and purify by ethanol precipitation. Refer to these publications:

- Babak T, Zhang W, Marros Q et al. (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10(11):1813-1819.
- Hiley SL, Jackman J, Babak T et al. (2005) Detection and discovery of RNA modifications using microarrays. Nucleic Acids Res 33(1):e2.
- Torchet C, Badis G, Devaux F et al. (2005) The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae. RNA 11(6):928-938.

Answer Id: E8095

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I used a mini-prep kit for propagating my lentiviral construct and obtained very low DNA yield. Can you please offer some tips?

Answer

The DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. Hence, we do not recommend using a mini-prep kit for propagation of lentiviral constructs. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or the 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: E9336

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I was not able to find a qPCR assay for my animal disease of interest. Do you offer custom assays?

Answer

Please contact Technical Support at animalhealthandfoodsafety@lifetech.com for further information.

Answer Id: E16420

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What types of molecules can be transfected with cationic lipid reagents?

Answer

Lipid reagents can be used to transfect DNA, RNA, oligonucleotides and siRNA. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. Lipofectin and Lipofectamine 2000 Reagent have also been used to transfect cells with proteins (Beta galactosidase and T3 polymerase).

Answer Id: E3128

Was this answer helpful?

Yes
No
Thank you for your response