Search Thermo Fisher Scientific
- Order Status
- Quick Order
-
Don't have an account ? Create Account
Search Thermo Fisher Scientific
Having difficulties with your experiment?
We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios.
View the relevant questions below:
Beginning your experiment?
Visit our
Storage at -80 degrees 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 degrees C or -80 degrees 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.
No, unfortunately not. Electrocompetent cells are not chemically treated. Unlike chemically competent cells, electrocompetent cells have an intact cell membrane.
To increase the intensity of the crystal violet-stained gel, allow the gel to lie for a couple of hours in crystal violet stain (45 µL of 2 mg/mL stain in 100 mL sterile water or 1-10 µg/mL stain in 0.1X TAE) and it will stain a little darker. Place the gel over a white background when excising the band to improve visibility.
The ends of pCR™II and pCR™2.1 vectors are palindromic sequences, since both ends must accommodate the TOPO® binding sites. Therefore, there is an inherent potential for secondary structure once the vector is ligated. This does not usually affect sequencing or PCR results, as cycling conditions are usually sufficient to overcome potential secondary structures that might form. However, certain inserts might enhance the formation of such secondary structures by the regions flanking the cloning site. Strong secondary structures may inhibit the cycle sequencing reactions commonly used in automated sequencing. If you suspect this is a problem for your clone, here are a few things to try:
Please try the suggestions below to increase the number of colonies.
Here are suggestions to try to increase your cloning efficiency with dTOPO® cloning:
Please consider the following possible causes:
No colonies may occur due to the following problems:
There could be a few possibilities for this:
On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.
You may be cloning in an artifact. TA and TOPO® cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
If the insert is potentially toxic to the host cells, here are some suggestions that you can try:
Here are possible causes and suggestions:
Phosphorylated products can be TA cloned but not TOPO® cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO® vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO® linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO® cloning.
Using the vector only for transformation is not a recommended negative control. The process of topo-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.
For Research Use Only. Not for use in diagnostic procedures.