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View additional product information for TOPO™ TA Cloning™ Kit, Dual Promoter, with One Shot™ MAX Efficiency™ DH5α-T1R E. coli - FAQs (K462001, K462040)
73 product FAQs found
We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.
We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.
The vector backbones for both of these vectors are very similar. The main difference is that the pCR4-TOPO vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR4-TOPO vector very useful for sequencing applications.
The vector backbones for both of these vectors are very similar. The main difference is that the pCRII-TOPO vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR2.1-TOPO vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.
The sequence of the control template is proprietary.
We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.
Equation:
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)
The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.
Equation:
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
Yes, the enzyme mix leaves 3' A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3' A-overhangs to the product for TA 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.
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.
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.)
There could be a few possibilities for this:
- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3' A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3' A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3' A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 µl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
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.
No colonies may occur due to the following problems:
Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). 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. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.
Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3' A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3' ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3' A next to another A. Taq is most efficient at adding a nontemplate 3' A next to a C. You may have to redesign your primers so that they contain a 5' G instead of a 5´ T.
Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 µL double diH2O to the 6 µL TOPO reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.
We would suggest using our TOPO TA cloning kit for sequencing, which contains the pCR 4 TOPO vector, or our Zero Blunt TOPO PCR cloning kit for sequencing, which contains the pCR4Blunt-TOPO vector.
Due to the small size of your product, we recommend using the pCR 2.1 TOPO vector for your cloning. This size fragment would not be able to fully interrupt the ccdB gene in the pCR4-TOPO vector, and therefore, you may not get colonies as ccdB is lethal to E. coli.
Regular TOPO TA Cloning kits are efficient for cloning PCR products up to approximately 2-3 kb. With PCR products larger than 3 kb, the efficiency of cloning drops significantly. The TOPO XL PCR Cloning Kit has been optimized for TOPO cloning of long (3-10 kb) PCR products.
If using the regular TOPO kits, here are some tips to improve efficiency:
1. Use crystal violet instead of ethidium bromide (EtBr) to visualize the PCR for gel isolation to avoid DNA nicks
2. Increase incubation time of the TOPO reaction to 30 mins
3. Keep insert:vector molar ratio low, optimally 1:1
4. Dilute reaction to 20 µL, while maintaining same amount of vector and insert. Increase the volume of the salt solution to 3.7 µL to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector ends.
Storage of the TOPO vector plus insert reaction for 1 week at 4 degrees C has shown no detectable decrease in the cloning efficiency of the TOPO reaction, as >95% of the colonies have insert. However, the total number of colonies was decreased by 10-fold. Storage of the TOPO reaction mix overnight at 4 degrees C showed little to no decrease in the number of colonies when compared to fresh TOPO reaction mix.
The composition of the 6X Stop solution is 0.3 M NaCl, 0.06 M MgCl2, and the composition of the 6X Salt solution is 1.2 M NaCl, 0.06 M MgCl2. Stop solution is only included in the TOPO XL Cloning kit whereas Salt solution is currently included in all of the other TOPO cloning kits. These solutions prevent free topoisomerase from re-binding and nicking the plasmid, which would reduce the number of colonies from a TOPO reaction.
When doing a TOPO cloning reaction, 2 µl of a PCR reaction containing up to 10% DMSO or 1.3 M betaine will not interfere with the TOPO reaction. Formamide and high levels of glycerol will inhibit the reaction. These reagents are usually added to the PCR reaction to enhance the yield of the PCR product, e.g., to reduce the effect of secondary structure or assist in amplification of GC-rich sequences. The effects of tricine or acetamide have not been tested on the TOPO cloning reaction.
PCR primers should not have 5'-phosphates when cloning into any TOPO vector, as the presence of 5'-phosphates inhibit the TOPO cloning reaction. 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 treated with phosphatase (CIAP) prior to TOPO-cloning. Treatment with CIAP may raise efficiency to 25%. PCR products generated with 5'-biotinylated primers (or any other 5'-label including 5'-Cy5) will not ligate into any of the TOPO vectors due to steric hindrance.
Gel purification is not required if the gel indicates that the PCR product is clean with no visible non-specific bands or primer dimers. It is recommended if the PCR product is >1.5 kb or if non-specific bands and primer dimers are visible on the gel. Smaller products clone much more efficiently into the vector than larger products; therefore, they should be eliminated from the sample prior to cloning. There is some reduction in A-overhangs if the PCR product is gel purified, which along with PCR product loss during the procedure may slightly reduce total number of colonies. However, the percentage of colonies with insert does not change; it is typically >90% recombinant clones.
For optimal TOPO cloning, we recommend using fresh PCR products.
TA cloning ligates the insert and vector using a T4 DNA ligase, while TOPO TA cloning uses the intrinsic properties of topoisomerase, which ligates the insert and vector during a 5 minute desktop reaction. TOPO TA cloning results in >95% recombinants, while TA cloning results in >80% recombinants.
We offer a custom service for TOPO cloning adaptation services. Our scientists can prepare your vector for either blunt TOPO cloning, TOPO TA cloning, or directional TOPO cloning of PCR products.
Yes, our pCR.1 TOPO TA (Cat. No. 450641), pCR4-TOPO TA (Cat. No. 450030), pCRBluntII-TOPO (Cat. No. 450245) are available separately.
No, we do not recommend this as these vectors contain the topoisomerase DNA protein complex conjugated to the end of the vector.
We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1 (insert:vector). The ng quantities should be between 5-10 ng of a 2 kb PCR product in a TOPO cloning reaction.
Please consider the following before TOPO cloning:
- TOPO cloning cannot ligate DNA with a 5' phosphate group.
- TOPO cloning will decrease in efficiency inversely with the size of the insert (above 3 kb) unless using the TOPO XL cloning kit.
- TOPO vectors contain different antibiotic resistance markers which should be considered before purchase.
- TOPO TA vectors accept fragments containing a 3' A overhang while Zero Blunt vectors accept fragments that are blunt-ended.
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.
Subcloning DH5? cells are a compatible strain, but you will get lower efficiency (10e6 vs 10e9) and therefore risk getting fewer clones. Top10F' is also compatible, but if blue/white screening is performed, IPTG along with X-gal will be needed for detection due to the expression of the lacIq repressor present in cells containing an F' episome.
Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.
Here are a few suggestions:
- Small fragments/linkers are cloning in to your vector instead of your insert; to correct this, gel-purify the insert before ligation
- Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used
- If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate
- Incubate your plate for a longer period to ensure full color development
While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.
With any strain, the first thing to try would be to lower the growth temperature of the culture to 30 degrees C or even lower (room temperature). Slower growth will generally allow E. coli to tolerate difficult sequences better. If reducing the growth temperature doesn't help, you may want to consider using a competent cell strain such as Stbl2 or Stbl4 cells, which have been shown to accommodate this type of sequence better than other strains in the same conditions.
We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.
The F' episome in TOP10F' has a lacIq marker, which over-expresses the lac repressor. IPTG must be added to LB plates along with X-gal to see beta-galactosidase expression and blue color in this strain. TOP10, on the other hand, does not require IPTG for blue/white screening.
There are a few conditions that can lead to this: SOC medium or other media used when plating was contaminated, DNA was contaminated with amp-resistant microbes, you used old plates with degraded amp, or the competent cells themselves were contaminated.
If you are using a mcr/mrr(+) competent cell strain, cellular enzymes may be recognizing eukaryotic methylation patterns on the yeast genomic DNA and deleting or rearranging it. Try a mcr/mr(-) strain such as Top10, DH10B, or OmniMAX 2.
XbaI cutting site is a Dam-methylation sensitive restriction site. TOP10 is a dam(+) strain, which means it expresses the methylating enzyme, Dam. You can try re-transforming into a dam(-) strain, such as INV110. Other dam- (and dcm-) sensitive restriction sites include the following:
- Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
- Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I
1. Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for ? complementation.
2. Select an E. coli strain that carries the lacZdeltaM15 marker.
3. Plate transformations on plates containing X-gal. Spread 50 µg of 2% X-gal or 100 microliters of 2% bluo-gal (both can be dissolved in DMF or 50:50 mixture of DMSO:water) on the surface of a 100 mm plate and let dry. Alternatively, add directly to the cooled medium (~50 degrees C) before pouring the plates at a final concentration of 50 µg/mL for X-gal and 300 µg/mL for bluo-gal. Plates are stable for 4 weeks at 4 degrees C.
4. If the strain used carries the lacIq marker, add IPTG to induce the lac promoter. Spread 30 µl of 100 mM IPTG (in water) on 100 mm plates. Alternatively, add the IPTG directly to cooled medium (~50 degrees C) before pouring the plates to a final concentration of 1 mM. Plates are stable for 4 weeks at 4 degrees C.
5. Do not plate E. coli on medium containing glucose if using X-gal or bluo-gal for blue-white screening. Glucose competes as a substrate and prevents cells from turning blue.
For long-term storage, preparation of glycerol stocks stored at -70 degrees C is recommended. Follow the protocol below:
1. Pick one colony into 5 mL LB broth or S.O.C. medium. Grow overnight at 37 degrees C.
2. Prepare glycerol solution: 6 mL of S.O.B. medium and 4 mL of glycerol.
3. Take one volume of cells and add one volume of glycerol solution and mix.
4. Freeze in ethanol/dry ice. Store at -70 degrees C.
Yes, this is possible. We recommend using saturating amounts of DNA (10 ng of each plasmid). Make sure that the origin of replication is different in each plasmid so that they can both be maintained in the cell. If the ori is the same, the plasmids will compete for replication and the one with even a slight disadvantage will be lost. Alternatively, cells with a resident plasmid can be electroporated with a second plasmid without “electrocuring” taking place.
In plates, we recommend 50 µg/mL X-gal and 1 mM IPTG (0.24 mg/mL). When spreading directly onto agar plates, we recommend 40-50 µl of 40 mg/mL X-gal (2% stock) in dimethylformamide and 30-40 µl of 100 mM IPTG on top of the agar. Let the X-gal and IPTG diffuse into the agar for approximately 1 hour. Do not plate on media containing glucose, as it competes with X-gal or bluo-gal and prevents cells from turning blue.
Competent cell efficiency is measured by transformation efficiency. Transformation efficiency is equal to the number of transformants, or colony forming units, per microgram of plasmid DNA (cfu/microgram).
Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.
DH5? cells are commonly used for routine cloning, but are mcr/mrr+, and therefore not recommended for genomic cloning. The TOP10 competent cells, on the other hand, contain mutated mcr/mrr, and therefore are a good choice for routine cloning and can be used for cloning of methylated DNA, such as eukaryotic genomic DNA. Our Mach1 strain is the fastest growing cloning strain that is T1 phage resistant.
These small colonies are most likely caused by degradation of the Ampicillin. The colonies are just untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase, the enzyme produced from the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.
One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.
Suggestions:
1. Use TOP10F' or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.
This may be caused by the instability of the insert DNA in TOP10 E. Coli. 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.
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.
If working with a vector that contains the lac promoter and the LacZ ? fragment (for ? complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid), it will repress expression from the lac promoter thus preventing blue/white screening. Hence, in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening.
TOPO vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. When doing blue/white color screening of clones in TOPO vectors containing the LacZ-ccdB cassette, colonies showing different shades of blue may be observed. It is our experience that those colonies that are light blue as well as those that are white generally contain inserts. The light blue is most likely due to some transcription initiation in the presence of the insert for the production of the lacZ alpha without enough ccdB expressed to kill the cells and is insert dependent. To completely interrupt the lacZ gene, inserts must be >400 bp; therefore an insert of 300 bp can produce a light blue colony. A white colony that does not contain an insert is generally due to a spontaneous mutation in the ccdB gene.
A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death. (Reference: Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.)
Strains that contain an F plasmid, such as TOP10F', are not recommended for transformation and selection of recombinant clones in any TOPO vector containing the ccdB gene. The F plasmid encodes the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do not contain the F plasmid. The CcdB protein is a potent cell-killing protein when the CcdA protein does not inhibit its action.
If working with a vector that contains the lac promoter and the LacZ alpha fragment (for ? complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid) it will repress expression from the lac promoter, thus preventing blue/white screening. Hence in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening. X-gal (also known as 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside) is soluble in DMSO or DMF, and can be stored in solution in the freezer for up to 6 months. Protect the solution from light. Final concentration of X-gal and IPTG in agar plates: Prior to pouring plates, add X-gal to 20 mg/mL and IPTG to 0.1 mM to the medium. When adding directly on the surface of the plate, add 40 µl X-gal (20 mg/mL stock) and 4 µl IPTG (200 mg/mL stock).
No, these vectors do not contain a functional promoter to express your gene of interest. These vectors are typically for subcloning or sequencing.
TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum Taq, Accuprime Taq, Platinum or Accuprime Taq High Fidelity, AmpliTaq, AmpliTaq Gold, or AmpliTaq Gold 360.
Blunt cloning:
- Use a proofreading enzyme such as Platinum SuperFi DNA Polymerase.
Directional TOPO cloning:
- Platinum SuperFi DNA Polymerase works well.
For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.
When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.
If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.
Some examples of Taq blends that are compatible with TOPO TA Cloning are Platinum Taq DNA Polymerase High Fidelity and AccuPrime Taq DNA Polymerase High Fidelity.
Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.
Yes, the TOPO TA cloning and Zero Blunt cloning vectors can be used for in vitro transcription. The T7, SP6, and T3 promoters contained in these vectors are active phage promoters, and the corresponding polymerases will transcribe RNA from these promoters.
These vectors are not generally recommended for in vitro translation because most of these vectors contain one or more translational initiation signals (ATG) downstream (3’) of the promoters contained in them, within the multiple cloning sites, which may interfere with attempts to translate an open reading frame in the insert.
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.
-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.
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.
Phosphorylated products can be TA-cloned, but not TOPO-cloned. This is because the necessary phosphate group is already contained within the Topoisomerase-DNA intermediate complex on the TOPO vectors. The TOPO plasmid has a 3' phosphate, to which Topoisomerase is covalently bound. The PCR product must provide 5'OH and 3'OH groups to react with the Topoisomerase, and presence of an additional phosphate group will inhibit the Topoisomerase-mediated ligation.
The non-TOPO TA Cloning vectors have a 3'OH and a 5' phosphate, and because they involve use of standard ligase, they are compatible with PCR products both with or without 5' and 3' phosphate groups.
Pre-phosphorylated PCR products can be phosphatase-treated (CIP/BIP) to remove the phosphate groups before TOPO cloning - however, cloning efficiency may be decreased compared with an insert that was never phosphorylated at all.
Here is a list of some commonly known PCR enzymes and indication of their compatibility with TA Cloning vectors.
1) Enzymes that leave abundant 3'-A overhangs, directly compatible with TA Cloning:
- Invitrogen Taq Polymerase, Platinum Taq Polymerase, Platinum PCR Super Mix
- Applied Biosystems AmpliTaq, AmpliTaq Gold, and AmpliTaq 360 DNA Polymerase
- Invitrogen SuperTaq and SuperTaq Plus
- Roche Taq Polymerase and Tth Polymerase
- TaKaRa Taq
- Agilent Taq2000 polymerase, Exo-Pfu Polymerase
2) Enzymes that leave partial A overhangs, generally contain mix of Taq and proofreader and may have lower efficiency with TA Cloning:
- Invitrogen Platinum Taq High Fidelity and Platinum PCR Supermix High Fidelity
- Roche Expand System
- TaKaRa LA Taq and Ex Taq
On a typical cloning plate with blue/white screening, there are a few white colonies which do not contain insert. These colonies 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). For the best chance to find a colony with a proper insert, pick clones of a variety of color and pattern for analysis. An insert may generate distinct patterns according to its orientation and size. Even dark blue colonies can have inserts present, if the insert is small and a resulting ORF is in frame with the LacZalpha.
Most of our cloning vectors are offered in a complete format, which includes competent cells. While in most cases other cells can be used with our vectors, we cannot guarantee the results you will get with our cloning vectors if you use your own competent cells. For this reason, most TOPO TA Cloning vectors can only purchased in a complete kit with cells, although there are a few exceptions. Non-TOPO vectors are generally available in multiple formats, with and without cells. To get the most current information on available products, visit the Cloning section of our website under Products & Service.
Zero Background and Zero Blunt vectors are available without competent cells provided, but you should especially careful in choosing competent cells to use with them. These vectors contain the ccdB gene for efficient negative selection of clones without insert, and some E. coli strains are not compatible with the mechanism of negative selection by the lethal activity of the ccdB gene product. In particular, cells with the F' episome have a ccd locus containing the ccdA gene, which prevents ccdB protein cell-killing. Therefore, cells without the F' episome are recommended so that only the CcdB protein will be expressed, and its cell-killing ability will not be inhibited or reduced by CcdA.
The pCRII-TOPO vector is a dual promoter vector. It contains a T7 promoter at the 5' end of the multiple cloning site and an Sp6 promoter at the 3' end. The pCR2.1-TOPO vector only contains the T7 promoter at the 5' end. The dual promoter vector is only of advantage for in vitro transcription studies with the cloned insert. In terms of cloning efficiency, there is no difference between the pCR2.1-TOPO and the pCRII-TOPO plasmids. Both the pCR2.1-TOPO and the pCRII-TOPO vector contain M13 sequencing primer sites to sequence the cloned insert in both directions.
TOP10, TOP10F', DH5a and INValphaF' are some of the strains offered with the Original TA Cloning Kits and the TOPO TA Cloning Kits. E. coli strains DH5a,TOP10, and INValphaF' do not have the lacIq repressor and permit constitutive expression from the lac promoter. With these cells, there is no need to add IPTG (inducer) for blue/white screening with X-gal. In contrast, E. coli strain TOP10F' carries the lacIq repressor and requires IPTG inducer to be added to enable blue/white screening. Please note, the F' episome in the INValphaF' strain is different from other strains in that it does not contain the lacIq repressor. Usually, presence of the F' and lacIq is only an advantage if you work with an insert that is potentially toxic to the host cell. If your insert is (potentially) toxic, we recommend using the TOP10F' cells without adding any IPTG. The lacIq repressor will repress expression from the lac promoter and you won't get blue-white screening, but you will still get colonies. This way you can clone a toxic construct.
TOPO TA cloning kits are also offered with Mach1 T1r competent cells. The Mach1 T1 Phage-Resistant (T1R) E. coli strain is the fastest growing chemically competent strain currently available. Doubling time is approximately 50 minutes, compared with an excess of 74 minutes for other cloning strains. Mach1 colonies are clearly visible within eight hours of plating the transformation mix, enabling you to plate and pick colonies in the same day. With these cells, there is no need to add IPTG (inducer) for blue/white screening.
The origin of replication in pCRII-TOPO is pUC-based.
The first ATG is contained within the M13 Reverse Primer sequence. Note that there is an additional ATG in frame just 6 bases further down.
The reading frame is as follows: GAA, TTC, GGC, TT., ..., etc. Therefore, 1 additional base is needed 5' to the reading frame of the gene of interest in order to be in frame with the LacZ gene.