Product FAQ

How does the pCR™4-TOPO™ and pCR™4Blunt-TOPO™ streamline sequencing?

Answer

Both the pCR™4-TOPO™ and pCR™4Blunt-TOPO™ vectors contain a minimized multiple cloning site. There are only 33 base pairs from the sequencing primer sites to the cloning site. This allows for more sequencing read of your insert and less of the vector which an save time analyzing the sequence data.

Answer Id: E3337

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Product FAQ

Is it possible to generate nested deletions using the TOPO™ Cloning Kit for Sequencing and the ZeroBlunt TOPO™ PCR Cloning Kits?

Answer

Yes. Both vectors (pCR™4 and pCR™-Blunt II) are designed to allow the creation of nested deletions for sequencing large inserts using the same sequencing primer. In addition, a comprehensive protocol is provided in the TOPO™ manual for these kits.

Answer Id: E3338

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Product FAQ

Why is there a TOPO™ Cloning kit "Stop Solution" mentioned in my manual, but it is not supplied in the TOPO™ Cloning kit?

Answer

Stop Solution is a reagent included in older versions of all TOPO™ kits, and still offered in the TOPO™ XL kits. It is very similar to the current TOPO™ cloning kit Salt Solution. Instruction manual versions preceding Version J recommended adding the Stop Solution at the end of the 5 minute TOPO™ reaction. The basic function of the Stop/Salt Solution is to keep the Topoisomerase from re-binding to the plasmid DNA and nicking the DNA. Later studies found that it was actually more effective to add this component during the TOPO™ cloning reaction versus afterward, and thus the Stop Solution was replaced with the similar but higher-concentration Salt Solution in most kits.

Answer Id: E3339

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Product FAQ

I’m having problems with my DNA solubilizing into solution when using TRIzol™ Reagent. What can I do?

Answer

The most common problem related to DNA solubilization occurs when the DNA pellets are overdried. It is very important not to dry pellets longer than 5 minutes. The use of vacuum suction devices to remove the wash solutions may cause overdrying of DNA pellets. Vacuum suction draws air through the pellet and almost always will overdry the DNA pellets. Avoid removing the wash solutions with any type of vacuum suction device and limit the drying time to <5 minutes. If you follow our simple recommendations below, you can avoid many nucleic acid solubility problems.

Remove droplets of ethanol from the wall of the test tube with a sterile cotton swab. Additional ethanol can be removed by touching the pellet with a sterile capillary pipette tip. Excess ethanol will be drawn inside the pipette by capillarity. Residual ethanol that may remain in the DNA pellet will not be harmful. You can usually eliminate DNA solubility issues by adding either TE buffer or 8 mM NaOH to the pellet before all of the ethanol has evaporated. The DNA pellets will become clear after a 5-10 minute incubation, as they begin to rehydrate. In order to solubilize the DNA completely, the solution must be pipetted up and down before removing an aliquot for quantitation.

DNA pellets that are overdried can be solubilized but it may be necessary to put them into the refrigerator and pipet them periodically until they become clear and go into solution.

Polysaccharides are water-soluble and they will partition into the aqueous phase with the RNA. Also, RNA and DNA pellets that contain contaminants tend to solubilize more easily than pellets that are very pure if they are not overdried. Pellets that do not solubilize in 8 mM NaOH will not solubilize in a phenol/chloroform solution, either.

Answer Id: E7713

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Product FAQ

What happens when a vector adapted with the Topoisomerase enzyme is stored at -80°C?

Answer

-80°C storage is fine. The TOPO™ vector can be freeze-thawed several times without loss of activity, and the vector is stable for storage at either -20°C or -80°C. However, note that the vector rapidly ages at room temperature (~15 min), which will result in a lower cloning efficiency (a reduction in the number of colonies following transformation). So during active use it should be kept on ice to prevent degradation, and should be quickly returned to the freezer.

Answer Id: E3340

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Product FAQ

What are the advantages of using TOP10 over DH5alpha cells for cloning?

Answer

The main advantage is that TOP10 cells have mutations in the mcrA, mcrB and mrr genes which encode restriction systems for methylated DNA. This means that you can clone highly methylated DNA derived from such sources as mammalian and plant cells, and it will not be degraded after transformation.

Answer Id: E3358

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Product FAQ

What tools are available to compare my data from the MG-U74v2 Set and the Mouse Expression Set 430?

Answer

There are multiple tools available for this type of exercise. The one you choose will depend on the nature of the comparison you'd like to do. To identify all probe sets, both old and new, that represent a particular transcript, we have the Probe Set Display tool, found on the NetAffx™ Analysis Center. In addition to do a more global comparison of probe set matches, there are comparison spreadsheets found on the Affymetrix™ web site.

Answer Id: E13724

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Product FAQ

I’m getting low yield of DNA/DNA degradation when isolating it with TRIzol™ Reagent. What happened?

Answer

Here are some possible causes for low yield/DNA degradation:

- The sample was not fully homogenized or lysed. If any solid material remains after chloroform is added, this indicates that DNA yield may be poor, as DNA will remain trapped in the unhomogenized material. You can remove unhomogenized material by passing the TRIzol™ homogenate (prior to phase separation) through a polypropylene filter cloth.
The final DNA pellet was not fully redissolved. It can take several hours to resuspend the DNA. Some incubation at 37 degrees C between pipettings will help. Also make sure that it is not too concentrated or it won't go back into solution. If the DNA is not fully redissolved, it will be lost during the final centrifugation when removing the gel-like material.
- The tissue was not IMMEDIATELY processed or frozen after removal from the animal or other source.
- Samples were homogenized with a high-speed homogenizer. DNA shearing can happen.
- If expected yield is <10 μg, there are limitations to the physical action of precipitation that would lead to low yields. A microcarrier (glycogen, tRNA) may be included in the homogenization and/or wash steps, or samples may be pooled to increase the expected yield.
- If you are only interested in quantitative DNA recovery, we suggest resuspending in freshly made 40 mM NaOH instead of 8 mM NaOH; this will hydrolyze DNA but will facilitate solubilization. Also, do not centrifuge after homogenization (prior to adding chloroform in the initial step), as some of the DNA will precipitate during this centrifugation. If the pellet is slippery, the speed of the first centrifugation may be increased to 5,000 x g; this will make the pellet more difficult to solubilize, and it may need to be vortexed and heated to 50 degrees C.

Answer Id: E7714

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Product FAQ

How do I know if I need heat-inactivated serum for my cells?

Answer

Usually the best way to find out is to go to the source you obtained your cells from. For example, ATCC will have serum requirements for the cells they sell. Most insect cell lines and embryonic stem cell lines require heat-inactivated serum.

Answer Id: E11878

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Product FAQ

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?

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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Product FAQ

Why is Beta-mercaptoethanol (BME) no longer included with the One Shot™ Chemically Competent E. coli kits? What was the purpose of the BME during E. coli transformation?

Answer

Beta-mercaptoethanol (BME) degrades carbohydrates on the cell surface, which theoretically allows DNA to get closer to the membrane prior to heat shock. In the past, this was thought to improve the efficiency of transforming E. coli strains, and the addition of Beta-mercaptoethanol was a standard practice for all chemical transformations. However, galU and galK minus strains, such as TOP10, INV?F', DH5?™, DH10B™ and TOP10F', have fewer carbohydrates on the cell surface. After repeated testing of all of our strains, we determined that adding BME had no beneficial effect on transformation efficiency, and we chose to remove BME from the chemically competent One Shot™ kits.

Answer Id: E3359

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Product FAQ

Is it possible to work with fewer cells than is specified for one test with Dynabeads™ FlowComp™ kits?

Answer

Most human FlowComp™ kit protocols are for 5 x 10E7 total cells or more per test. You can work with fewer cells, but we don't recommend that you scale down the volume of Dynabeads™ magnetic beads or the buffers; stick to the volumes recommended for 5 x 10E7 cells. This is also true for the release step, where the volume of release buffer must be at least 500 µL, so make sure not to scale down more than recommended.

Answer Id: E12111

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Product FAQ

Where can I obtain sequence information for the probes and probe sets represented on the arrays in the GeneChip™ Mouse Expression Set 430?

Answer

Complete sequence information for the probes, target, and consensus/exemplar sequences represented on all catalog expression arrays, including the Mouse 430, are available through the Support by Product page.

Answer Id: E13725

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Product FAQ

I’m getting low absorbance for DNA (A260/A280) ratio <1.70 after TRIzol™ Reagent extraction. What could be causing this?

Answer

Typically, low absorbance is due to phenol contamination. You should include additional washes with 0.1 M sodium citrate in 10% ethanol. It's not unusual for residual phenol from the extraction to remain, and the A260/A280 ratio of the extracted material would show a higher than expected A280. We recommend a second ethanol precipitation to remove remaining phenol. This will also remove any excess salt. If the tube smells like phenol after the procedure is done, precipitate the DNA again. It is important to do this, as phenol inhibits downstream enzymatic reactions.

Answer Id: E7715

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Product FAQ

Is it true that phenol red has estrogenic properties?

Answer

Phenol red in tissue culture is a weak estrogen. Implications concerning study of estrogen-responsive cells in culture are discussed in Proc Natl Acad Sci U S A 83:2496 (1986).

Answer Id: E11879

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