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FAQ
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.5 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences.
How should I store my Supreme NZYTaq II 2× Green Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
Supreme NZYProof 2x Colourless Master Mix
The master mix should be stored at -20 ºC, in a constant temperature freezer. Minimize the number of freeze-thaw cycles by storing in working aliquots. The Mix maybe stored at 4 °C for up to 7 days.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
How should I store my NZYProof 2x Green Master Mix?
The master mix should be stored at -20 ºC, in a constant temperature freezer. Minimize the number of freeze-thaw cycles by storing in working aliquots. The Mix maybe stored at 4 °C for up to 7 days.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
How should I store my NZYProof 2x Colourless Master Mix?
The master mix should be stored at -20 ºC, in a constant temperature freezer. Minimize the number of freeze-thaw cycles by storing in working aliquots. The Mix maybe stored at 4 °C for up to 7 days.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Template DNA damaged or degraded. An intact, high-quality template is essential to achieve a reliable amplification of large fragments. Extreme care must be taken in the preparation and handling of DNA. Always use purified high-quality DNA as template.
d) Concentration of magnesium is too low . Mg2+ is included in the 10× Reaction Buffer at a final concentration of 2 mM, which is sufficient for most targets. For some targets, a higher Mg2+ concentration may be required. Titrate from 2 mM to 4 mM (final concentration) in 0. 5 mM increments.
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Check the quality and concentration of primer solutions. We recommend to prepare small-volume working aliquots from the stock solution. Avoid using primers subjected to multiple freezing-thawing cycles.
How should I store my Supreme NZYLong DNA polymerase?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Template DNA damaged or degraded. An intact, high-quality template is essential to achieve a reliable amplification of large DNA fragments. Extreme care must be taken in the preparation and handling of DNA. Always use purified high-quality DNA as template.
d) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.5 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Check the quality and concentration of primer solutions. We recommend to prepare small-volume working aliquots from the stock solution. Avoid multiple freeze cycles.
How should I store my NZYLong 2× Colourless Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Template DNA damaged or degraded. An intact, high-quality template is essential to achieve a reliable amplification of large DNA fragments. Extreme care must be taken in the preparation and handling of DNA. Always use purified high-quality DNA as template.
d) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.5 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Check the quality and concentration of primer solutions. We recommend to prepare small-volume working aliquots from the stock solution. Avoid multiple freeze cycles.
How should I store my Supreme NZYLong 2× Colourless Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Template DNA damaged or degraded. An intact, high-quality template is essential to achieve a reliable amplification of large DNA fragments. Extreme care must be taken in the preparation and handling of DNA. Always use purified high-quality DNA as template.
d) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.5 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Check the quality and concentration of primer solutions. We recommend to prepare small-volume working aliquots from the stock solution. Avoid multiple freeze cycles.
How should I store my Supreme NZYLong 2× Green Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature/ primer concentration. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (3 ° to 5 °C lower than Tm). Optimise annealing temperature using temperature gradient PCR. Increase primer concentration for lower yield amplicons.
b) Inadequate cycling conditions. Increase extension time and/or number of cycles.
c) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
d) Inadequate amount of template DNA. Titrate template amount. We recommend starting with 20-50 ng of genomic DNA.
c) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.25 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another pool of primers, avoiding complementary sequences. Ensure that each primer pair originates a single product without primer-dimers in individual PCR reactions. If necessary, reduce primer concentration. Reduce the number of cycles.
What is the maximum size of amplicon using Multiplex PCR NZYTaq 2x Green Master Mix?
Multiplex PCR NZYTaq 2x Green Master Mix allows efficient multiplexing of targets ranging in size from 70 bp to 2.5 k.
Which type of DNA ends are generated in the PCR products using Multiplex PCR NZYTaq 2x Green Master Mix?
Resulting PCR products have an A-overhang and are suitable for cloning with NZYTech´s NZY-A PCR cloning kit (MB053) or NZY-A Speedy PCR cloning kit (MB137).
I don’t distinguish separate bands using agarose gel electrophoresis. What should I do?
When separating samples through agarose gel electrophoresis, please take into account that both concentration and grade of agarose are important when working with closely-sized fragments. NZYTech offers a vast portfolio of agaroses with different specifications to cover a wide range of needs. Please visit www.nzytech.com/products-services/category/molecular-biology/products/agaroses/ to identify the agarose that most suits your experiment.
How should I store my Multiplex PCR NZYTaq 2x Green Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature/ primer concentration. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (3 ° to 5 °C lower than Tm). Optimise annealing temperature using temperature gradient PCR. Increase primer concentration for lower yield amplicons.
b) Inadequate cycling conditions. Increase extension time and/or number of cycles.
c) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
d) Inadequate amount of template DNA. Titrate template amount. We recommend starting with 20-50 ng of genomic DNA.
c) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.25 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another pool of primers, avoiding complementary sequences. Ensure that each primer pair originates a single product without primer-dimers in individual PCR reactions. If necessary, reduce primer concentration. Reduce the number of cycles.
What is the maximum size of amplicon using Multiplex PCR NZYTaq 2x Colourless Master Mix?
Multiplex PCR NZYTaq 2x Colourless Master Mix allows efficient multiplexing of targets ranging in size from 70 bp to 2.5 k.
Which type of DNA ends are generated in the PCR products using Multiplex PCR NZYTaq 2x Colourless Master Mix?
Resulting PCR products have an A-overhang and are suitable for cloning with NZYTech´s NZY-A PCR cloning kit (MB053) or NZY-A Speedy PCR cloning kit (MB137).
I don’t distinguish separate bands using agarose gel electrophoresis. What should I do?
When separating samples through agarose gel electrophoresis, please take into account that both concentration and grade of agarose are important when working with closely-sized fragments. NZYTech offers a vast portfolio of agaroses with different specifications to cover a wide range of needs. Please visit www.nzytech.com/products-services/category/molecular-biology/products/agaroses/ to identify the agarose that most suits your experiment.
How should I store my Multiplex PCR NZYTaq 2x Colourless Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature/ primer concentration. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (3 ° to 5 °C lower than Tm). Optimise annealing temperature using temperature gradient PCR. Increase primer concentration for lower yield amplicons.
b) Inadequate cycling conditions. Increase extension time and/or number of cycles.
c) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
d) Inadequate amount of template DNA. Titrate template amount. We recommend starting with 20-50 ng of genomic DNA.
c) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.25 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another pool of primers, avoiding complementary sequences. Ensure that each primer pair originates a single product without primer-dimers in individual PCR reactions. If necessary, reduce primer concentration. Reduce the number of cycles.
What is the maximum size of amplicon using Multiplex PCR NZYProof 2x Colourless Master Mix?
Multiplex PCR NZYColourless 2x Green Master Mix allows efficient multiplexing of targets ranging in size from 70 bp to 2.5 k.
Which type of DNA ends are generated in the PCR products using Multiplex PCR NZYProof 2x Colourless Master Mix?
Resulting PCR products have blunt-ends and are suitable for cloning with NZYTech´s NZY-blunt PCR cloning kit (MB121).
I don’t distinguish separate bands using agarose gel electrophoresis. What should I do?
When separating samples through agarose gel electrophoresis, please take into account that both concentration and grade of agarose are important when working with closely-sized fragments. NZYTech offers a vast portfolio of agaroses with different specifications to cover a wide range of needs. Please visit www.nzytech.com/products-services/category/molecular-biology/products/agaroses/ to identify the agarose that most suits your experiment.
How should I store my Multiplex PCR NZYProof 2x Colourless Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Additives required. Adding PCR-enhancing agents, like NZYTaq 5x Optimizer Solution (MB060) or NZYTaq 2x GC-Enhancer solution (MB143) may improve yield while amplifying difficult templates.
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Adjust concentration of magnesium. Generally, 2-3 mM MgCl2, typically 2.5 mM final concentration, works well for the majority of PCR reactions. Optimal concentration depends on target template, buffer and dNTPs. Optimise magnesium concentration by supplementing MgCl2 in 0.5 increments up to 4 mM.
How should I store my NZYTaq II DNA polymerase?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Inadequate PCR Setup. Adjust the primers concentration. Be sure to add the DNA template in last, and that you prepare the PCR mix at room temperature.
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Adjust concentration of magnesium. Generally, 2-3 mM MgCl2, typically 2.5 mM final concentration, works well for the majority of PCR reactions. Optimal concentration depends on target template, buffer and dNTPs. Optimise magnesium concentration by supplementing MgCl2 in 0.5 increments up to 4 mM.
How should I store my Supreme NZYTaq II DNA polymerase?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Concentration of magnesium is too low. Mg2+ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg2+ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.5 mM increments. (Note: MgCl2 is not provided in separate tubes).
How can I reduce the number of non-specific bands?
Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences.
How should I store my NZYTaq II 2× Colourless Master Mix?
The product should be stored at -20 ºC, in a constant temperature freezer.
What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
How should I store my Speedy NZYProof DNA polymerase?
The product should be stored at -20 ºC, in a constant temperature freezer.
I’d like to clone a fragment amplified with NZYSpeedy Proof DNA polymerase. How should I proceed?
Speedy NZYProof DNA polymerase has 3´→ 5´ exonuclease activity and will generate blunt-ended products. So, blunt-end cloning is recommended, by using NZY-blunt PCR cloning kit (MB121) or other similar cloning kit. Nevertheless, if TA cloning is required, the generated blunt-ended PCR fragments can be modified by adding 3´A-overhangs with a different polymerase, such as Taq DNA polymerase (see the A-tailing protocol in the product brochure of NZY-A PCR cloning kit – MB053). Due to the strong proofreading activity of Speedy NZYProof DNA polymerase, we recommend immediately proceed with cloning after this procedure, in order to avoid lost of the 3´A-overhangs during storage. Alternatively, the PCR products can be cloned using the innovative NZYEasy Cloning & Expression Systems in a rapid, precisely and ligase-independent manner. For optimal cloning efficiencies, silica-column purification of the PCR product using NZYGelpure kit (MB011) or other similar kit is highly recommended. This procedure will remove dNTPs, unused primers and other impurities. Spin-column purification is sufficient as long as the product is >90% pure. Gel-extraction should be performed in case non-specific amplifications and/or primer-dimers are formed.
What are the benefits of this method compared to classical cloning methods?
NZYEasy Cloning & Expression System relies on a simple reaction that enables the assembly of DNA inserts into a cloning or an expression vector in a precise and predetermined order. No insert phosphorylation, blunt-end polishing or digestion is required, thus making the NZYEasy Cloning & Expression System fast when compared to classical cut and ligation methods. Furthermore, cloning can be achieved either directly using blunt-ended or Taq-generated PCR products. The cloning is performed in a single reaction with high efficiencies compared to traditional methods, even when simultaneously clone multiple inserts into one vector.
Is it necessary to purify PCR products?
Spin-column purification of PCR products is highly recommended to remove dNTPs and impurities resulting from the amplification. Presence of non-specific amplification products and/or primer-dimers requires gel-purification of the amplified nucleic acid, which will enhance cloning efficiencies.
To amplify the insert, do I need to use PCR primers that have been purified by PAGE or HPLC?
No, you can use standard, desalted primers.
The insert will be cloned in reading-frame into pHTP vectors?
Yes. Inserts correctly cloned into pHTP1 expression vector will maintain reading frames starting on the ATG codon.
What type of competent cells is suitable to transform DNA clones obtained from the ligase-independent cloning reaction?
We recommend using E. coli DH5α (NZYTech, cat. No. MB004) as a host for cloning. Regarding expression, all E. coli strains expressing the gene that codifies for T7 RNA polymerase, such as BL21(DE3) (NZYTech, cat. No. MB006), are suitable to use.
Can other vectors be used instead of pHTP vectors?
No. pHTP vectors were designed to contain the required insert-complementary overhangs generated by PCR, as well as the specific sequence recognized by the NZYEasy enzyme mix. In theory, any vector can be converted into a “pHTP vector” to function with the NZYEasy Cloning & Expression System. In case vectors available in our pHTP portfolio do not fit your needs, please contact us at services@nzytech.com. Solubility tags can easily be included in the pHTP1 backbone and other vectors can be engineered to be used with this system. The NZYTech R&D team is available to assist you in case of need.
It is possible to clone many different inserts into one vector?
Yes, the NZYEasy Cloning & Expression System allows multiple fragment cloning into one pHTP vector. DNA fragments will be correctly annealed together in the desired order on the pHTP vector through the action of the highly efficient NZYEasy enzyme mix, free of any DNA ligase. Just combine the PCR-generated fragments with appropriate complementary overhangs, by allowing assembling in the order you choose, and a linearized pHTP vector. Both first and last inserts should have overhangs complementary with respective ends of the vector, thereby allowing efficient cloning. Each DNA fragment, encoding for an individual domain of the final multidomain construct, are kept in frame and are separated from each other by a linker sequence of 6 amino acids.
How many DNA fragments can be cloned in one reaction?
The number of inserts that can be joined in the same vector depends on their length and sequence. NZYEasy Cloning & Expression System has been tested efficiently for cloning 5 different genes of around 500-700 bp in size in the same vector. Note that the cloning efficiency decreases as the size of the final construct increases. However, if the simultaneous cloning of the desired inserts does not work, you can try a sequential cloning approach.
How can I reduce the number of false positive colonies containing the vector-backbone only?
Gel-purify the PCR band of interest in order to remove the template plasmid. Alternatively, digest PCR products with DpnI (NZYTech cat No. MB078) restriction enzyme to eliminate parental methylated DNA templates. When possible, you can also reduce the amount of template plasmid DNA to 0.1-0.5 ng per 50 µL PCR reaction.
The cloning reactions originate no colonies or a few number of colonies. What should I do?
Perform a cloning reaction using the PCR fragment provided as positive control with NZYEasy Cloning & Expression kits. Using this procedure, you can evaluate the functionality of cloning and transformation conditions.
If cloning and transformation result successfully using the positive control, then make sure that your DNA insert and the linearized pHTP vector share the required end-terminal complementarity by analysing primer sequences; verify the quality of the insert by gel electrophoresis; optimize the ligase-independent cloning reaction trying different vector:insert molar ratio, and/or repeat PCR and gel-purify the PCR product for a new cloning reaction and transformation. Alternatively, consider that the cloned insert may be toxic to E. coli. If this is the case, and if you are directly cloning into the expression vector, try to clone first into the low-copy number pHTP0 vector.
If cloning and transformation do not result successfully, check also the transformation efficiency of E. coli competent cells; verify if LB plates contain the appropriate antibiotic for the pHTP vector you are using; make sure you correctly handle the NZYEasy enzyme mix; verify if a specific component is missing in the cloning reaction.
Why should I use NZYTech’s gene synthesis services?
– You can focus on your research while we produce your gene
– It is possible to codon optimize your gene, thus increasing the possibility of achieving high protein levels
– Gene synthesis provides access to DNA templates that are not easily available on nature
Is it possible to synthesize sequences with high/low GC content or repeats?
Yes, NZYTech successfully synthesizes complex genes.
Is it possible to optimize gene design for high levels of expression?
Yes, ATGenium allows optimizing not only critical factors in transcription and translation but also factors related with co-translational protein folding. ATGenium comprises an extensive collection of codon usage tables, allowing optimizing codon usage for improved protein expression in different host systems.
Are all genes sequence-verified?
All genes are 100% sequence verified.
In which vector am I going to receive my gene?
You will receive your gene cloned into one of our standard cloning vectors with resistance to Ampicillin or Kanamycin.
Is it possible to sub-clone my gene into an expression vector?
Yes, your synthetic gene can be easily transferred from the entry vector to any of our pHTP E. coli expression vectors for a very competitive price.
How do you deliver synthetic genes?
NZYTech delivers 0.5 to 1 µg of plasmid DNA.
What is NZYTech’s policy for Intellectual Property Protection?
NZYTech does not claim rights or ownership of any intellectual property related to the DNA/amino acid sequences provided by our customers or the resulting synthetic genes. No data or material will be released to a third party without your consent. All genes resulting from gene synthesis and/or cloning services are stored at NZYTech for a three months period. After this period nucleic acids provided to customers are discarded.
Why should I use NZYTech’s gene synthesis services?
– You can focus on your research while we produce your gene
– It is possible to codon optimize your gene, thus increasing the possibility of achieving high protein levels
– Gene synthesis provides access to DNA templates that are not easily available on nature
Is it possible to synthesize sequences with high/low GC content or repeats?
Yes, NZYTech successfully synthesizes complex genes.
Is it possible to optimize gene design for high levels of expression?
Yes, ATGenium allows optimizing not only critical factors in transcription and translation but also factors related with co-translational protein folding. ATGenium comprises an extensive collection of codon usage tables, allowing optimizing codon usage for improved protein expression in different host systems.
Are all genes sequence-verified?
All genes are 100% sequence verified.
In which vector am I going to receive my gene?
You will receive your gene cloned into one of our standard cloning vectors with resistance to Ampicillin or Kanamycin.
Is it possible to sub-clone my gene into a commonly used expression vector?
Yes, we can sub-clone your gene into your vector of choice. Alternatively, your synthetic gene can be easily transferred from the entry vector to any of our pHTP E. coli expression vectors for a very competitive price.
How do you deliver synthetic genes?
NZYTech delivers 0.5 to 1 µg of plasmid DNA.
What is NZYTech’s policy for Intellectual Property Protection?
NZYTech does not claim rights or ownership of any intellectual property related to the DNA/amino acid sequences provided by our customers or the resulting synthetic genes. No data or material will be released to a third party without your consent. All genes resulting from gene synthesis and/or cloning services are stored at NZYTech for a three months period. After this period nucleic acids provided to customers are discarded.
Is it possible to optimize gene design for high levels of expression?
Yes, ATGenium allows optimizing not only critical factors in transcription and translation but also factors related with co-translational protein folding. ATGenium comprises an extensive collection of codon usage tables, allowing optimizing codon usage for improved protein expression in different host systems.
Why should I use NZYTech’s gene synthesis services?
– You can focus on your research while we produce your gene
– It is possible to codon optimize your gene, thus increasing the possibility of achieving high protein levels, and increasing the possibility of obtaining a non-complex sequence (compatible with Gene Fragments synthesis)
– Gene synthesis provides access to DNA templates that are not easily available on nature
Is it possible to synthesize gene fragments with high/low GC content or repeats?
No, NZYTech only synthesizes non-complex gene fragments. If you intend to synthesize a complex gene NZYTech can offer you the Custom Gene Synthesis service.
Are Gene Fragments sequence-verified?
No, Gene Fragments quality control procedure only involves size verification by electrophoresis. NZYTech’s Gene Fragments are synthesized under the same careful conditions as our custom gene synthesis service. However, since Gene Fragments are delivered to you as uncloned products the existence of minor populations of mutated or truncated fragments is inevitable.
In which vector am I going to receive my gene fragment?
Gene fragments are delivered as uncloned products.
How do you deliver synthetic gene fragments?
NZYTech delivers 100 ng to 1000 ng of dry dsDNA fragment. If you order more than 10 fragments NZYTech will deliver all fragments in a 96-well plate. Otherwise, each fragment will be delivered in tube.
What is NZYTech’s policy for Intellectual Property Protection?
NZYTech does not claim rights or ownership of any intellectual property related to the DNA/amino acid sequences provided by our customers or the resulting synthetic genes. No data or material will be released to a third party without your consent. Gene fragments produced are not stored in-house after dispatch to clients.
What payment methods can I use?
You can use Credit Card, Visa, MasterCard, American Express, Maestro, iDeal, Paypal or Apple Pay
How long will it take to receive my order?
It will take about 3 to 5 days depending on your location.
Do we deliver our products on weekends?
Depending on the shipping method selected on the order.
How will I receive my new Veloretti?
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How much does the shipping cost?
It's going to depend on the location.
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What is your return policy?
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Where can I find more product details?
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What is my warranty?
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Are all job opportunities listed on the website?
Yes, all currently active employment opportunities are listed on this website
What is the best way to apply for a position?
It is strongly recommend that you apply using the online form available on this page.
I applied for an available position. What are the next steps?
When you apply on-line for a specific position, you will receive a confirmation via email, and the information you prvided becomes available to our Human Resources Department. Qualified applicants will then be contacted by our Human Resources representative or the hiring manager
Can we request samples?
NZYTech can provide samples after the definition of customer needs and feasibility of product is confirmed.
Does NZYTech offer lyophlized products?
Yes, NZYTech can provide a broad range of lyo-ready or lyophilized product, both off-the-shelf selection or for a customized formulations
Are there additional fees, such as licensing?
NZYTech does not add any licensing fees. Quotes are provided as a one-off total sum and are note dependent on continued use after delivery.