Technical FAQs

pOnebyOne™ is a versatile Bicistronic Mammalian Expression family Kits. All vectors include 2A sequence to produce stoichiometric amounts of a protein of interest and a reporter protein. 2A is a valuable short element that allow to check the production of our protein of interest via a reporter protein. Three reporter proteins could be used: truncated nerve growth factor receptor (ΔNGFR) for cell membrane location or green fluorescent protein (GFP) and firefly luciferase (LUC) for intracellular location. Two strong promoters could drive the expression of both genes: cytomegalovirus or elongation factor 1 alpha promoter. pOnebyOne™ vectors are available with Neomycin, Puromycin or Hygromycin resistance cassettes to stable transfection. Viral vectors are also included in it.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

There are 26 vectors available. You can choose the most convenient pOnebyOne™ Kit using visiting pOnebyOne™ Bicistronic Mammalian Expression Kits Selection Guide.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

pOnebyOne™ vector family offers a complete solution to express your gene of interest because the kits include a linear vector to directional cloning of  PCR fragment and an efficient bicistronic expression system. pOnebyOne™ Mammalian Expression System is a wise choice because of:

• It allows directional cloning of PCR with the gene of interest.
• It does not require the use of restriction enzymes.
• Low background.
• Highly efficient cloning system tested with up to 4 kb inserts.
• It has got bicistronic expression cassette which enables stoichiometric production of reporter protein and protein of interest.
• Integral solution: a directional cloning vector to clone and produce a protein of interest.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Of course. There are vectors with Neomycin, Puromycin or Hygromycin resistance cassettes included in pOnebyOne™ Kits. All posible combinations of promoter and reporter proteins are available.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

• The volume of T4 DNA Ligase used is higher than 1/10 of the final reaction volume.
• The volume of ligated DNA was higher than 20 % of the competent cell volume.
• The ligated DNA was degraded due to a contaminating nuclease in the water or other reagents used in the ligation before transformation.
• Ligation failed because there was no ATP or Mg²⁺: we suggest use the supplied buffer. The ATP in buffer older than one year or buffer with multiple freeze-thaw cycles may have degraded enough to cause problems. So, we advise you make aliquots of the buffer.
• The ligation failed due to high salt or EDTA in the reaction: we recommend to clean up the DNAs.
• The restriction enzymes or phosphatase used for the dephosphorylation step are still active. Remove them using a clean up step.
• We advise to use PEG 6000 at 5 % in the reaction mixture if the ligation is between single base overhang molecules or blunt ended molecules.
• The insert and the plasmid do not have phosphates at the ends; we suggest using pSpark® DNA Cloning  if you have to clone a dephosphorylated blunt fragment. In other hand, you must phosphorylated at least one DNA to be joined.
• The ligase was inactive: we recommend to test on lambda HindIII or other convenient substrate.
• Check the compatibility of the ends to be joined.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

No, is not necessary to purchase phosphorylated primers or phosphorylate PCR product. Any primer you already have in your lab can be used for cloning into pSpark® TA and pSpark® TA Done DNA cloning vector. Phosphorylated primers do not inhibit ligation and thus can also be used. There is no need to add any specific sequence at 5 ́-ends of primers for cloning into pSpark® TA DNA Cloning.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

You should be use the following control reactions:

• Transformation efficiency depends largely on the cell competence. Test transformation efficiency of the competent cells (like CVX5α™ Chemically Competent Cells) using a supercoiled vector. Perform a transformation reaction and plate the number of cells that is expected to generate 50-100 colonies per plate, based on the anticipated transformation efficiency of the competent cells. The expected number of colonies should be seen, indicating that the competent cells were transforming with high efficiency. If the expected number of colonies is not seen, please check your competent cells and/or transformation protocol. To transform ligation mixtures, cell competence around 1×10⁷ CFU/ ug DNA allows optimal results.
• Religated vector as control. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Religation of vector with cohesive ends should result in less than or equal to 50% of the number of colonies obtained with supercoiled vector DNA, indicating that the components of the ligation reaction are working; religation of vectors with blunt ends should yield 10% to 20% of the number of colonies obtained with supercoiled vector DNA. This is an appropriate control only with vectors that have been digested with a single restriction endonuclease or endonucleases that generates compatible ends.
• Transformant cells are not viable in the selection plate used.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

The choice of a DNA Polymerase must be based on your goals.

Horse-Power™ Taq DNA Polymerase is a robust and cheap enzyme for routine amplifications. It is the enzyme to choose wether the cost per reactions is a priority or if you are using the amplified DNA to clone into a 3´T overhang vector; can not be cloned into a blunted vector (such as pSpark® I DNA Cloning Vector) a PCR fragment amplified with Taq DNA polymerase. Also, take in account that Taq polymerase is not a proofreading enzyme with an error rate around 2×10^-5 errors per nt per cycle.

But if you have planned to carry out experiments with Recombinant Proteins, expressed in prokaryotic or eukaryotic systems, we highly recommended to use High Fidelity Polymerases (like FastPANGEA™ High Fidelity DNA Polymerase). Keep in mind  this enzymes produces blunt-end fragments that can not be cloned , without prior end treatment, in the known T – vectors.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

• All pSpark® DNA cloning vectors have Ampicillin resistance for selection of transformants.
• All inserts can be amplified by PCR with pUC/M13 forward and reverse primers and in vitro transcribed with either T7 or SP6 RNA polymerases.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

You should use the following control reactions:

• Restriction endonuclease-digested vector. Perform a transformation with an amount of restriction enzyme digested vector  equivalent to that contained in the fraction of the ligation reaction being used for the experimental transformations. Do not use T4 DNA Ligase. Few or no colonies should be seen, indicating complete restriction endonuclease digestion of the vector. The presence of colonies indicates incomplete digestion of the vector that will cause a background of colonies containing non-recombinant vector in the experimental transformations.
• Restriction endonuclease-digested, dephosphorylated, religated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Few or no colonies should be observed, indicating complete desphosphorylation of the vector. A dephosphorylated vector should not be re-ligated by T4 DNA Ligase presence.
• No DNA transformation control. Perform a mock transformation of competent cells, to which no DNA is added. No colonies should be seen, indicating that the selection Antibiotic on the agar plates is potent and that the competent cells are pure.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

In general, the final concentration may vary between 1 and 3 mM when using 0,2 mM dNTPs (like TruePure™ dNTPs). EDTA or excess dNTPs can inhibit amplification by chelating the Magnesium ions necessary for Taq DNA Polymerase activity (like Horse-Power™ Taq DNA Polymerase).

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Yes. We provide with our vectors a highly active T4 DNA Ligase with the lowest nuclease activity. We highly recommend using it to get the maximum cloning efficiency. Please check both the total number of white colonies and the ratio of white positive colonies to blue negative colonies using the supplied Control Insert. If you obtain less than 700 white colonies (using cells with 2 x 10⁷ cfu/μg) and more than 4% of blue colonies then maybe our T4 DNA Ligase can help you in your laboratory.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

ATP is necessary for enzymatic function. It is involved in phosphorylating the ligase prior to the ligation reaction. Ligation efficiency is markedly reduced by removing ATP from the reaction. It is important, therefore, to handle the buffer appropriately in order to minimize degradation of ATP.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Horse-Power™ Taq DNA Polymerase is an isolated enzyme from the thermophilic bacteria Thermus aquaticus. It operates at different temperatures with varying efficiency: at 72 ºC 100% efficiency, at 85 ºC and 55 ºC has 40 % efficiency, at 37 ºC has a 2,5 % of efficiency and at room temperature about 0,4 %. Native and recombinant enzyme are termostable at high temperature: half-life greater than 2 hours at 75-80 ºC, 40 minutes at 95 ºC. The most common PCR programs employa denaturing temperature of 94 ºC and elongation temperature of 72 ºC.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

None of pSpark® DNA Cloning Kits are mixed or bounded to any protein.  By an original procedure, vector ends are treated to prevent recircularization. The result is a low background and highly stable DNA vector that could be even shipped at Room Temperature (ever ligase is absent: only samples and kits without ligase).

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

At least one molecule in the ligation reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5′ phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (not dephosphorylated) is routinely performed; not so if you ligate a dephosphorylated vector with a PCR amplified fragment using non phosphorylated primers. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

The Annealing Temperature to use in a PCR program is based on the value of theoretical Tm (Tm, melting temperature). This value is the temperature at which 50 % of the primer and its complementary sequence are present in a duplex DNA molecule. The Tm optimal range is between 50 ºC to 70 ºC and it can be determined by most computer programs which include mathematical formulas that due to different considerations (% GC, NaCl or Mg²⁺ concentration).

The best way to find practical Tm is running gradient PCR; many times this optimized Tm value is optimal in gradiente machine but not in other PCR machines. If your laboratory do not have one,  the annealing temperature of PCR program initially set based on 3-5 °C below to the calculated Tm.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

For cloning into pSpark® TA and pSpark® TA Done vector any non-proofreading DNA Polymerase could be used. Note that blend polymerases are composed, in a high percentage, of non-proofreading DNA Polymerase. All non-proofreading DNA Polymerases generates PCR fragments with 3´-adenine overhangs. Blend polymerases produces a mixture of PCR fragments: about 70 % fragments with 3′ -adenine overhangs and 30% of blunt-end fragments.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

For cloning an insert with one cohesive end and the other, blunted, use T4 DNA Ligase protocol for blunt ends. The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. Therefore you should use the more stringent protocol for blunt-end ligation.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

• Design primers 15-30 nucleotides in length.
• Avoid complementarity between the primers to prevent primer-dimer formation.
• Avoid inverted repeats (self-complementarity).
• GC-content of the primer should be around 40-60 % and these nucleotides should be dstributed uniformly avoiding stretches of them.
• Melting temperature of a pair of primer should not differ by more than 5 ºC.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Ligation reagents may be tested by performing a ligation reaction with a molecular size marker such as 1 kb DNA Ladder or Lambda DNA/Hind III Fragments. Compare the ligation reaction products to unligated DNA on an agarose gel. The ligation reaction should contain a high molecular weight smear and few low-molecular weight bands.

If the marker ligation does not work, use a fresh ligase. Another reason for low activity could be degradation of the ATP in the reaction buffer; use 5X Ligase Buffer that is less than 24 months old. The buffer should be stored at -20 °C, preferably in aliquots.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

• Check your primers do not bind to another region of the template DNA.
• Make sure primers don´t have complementary at the 3´ ends.
• Optimize the annealing step by increasing the temperature  in 2-5 ºC.
• Optimize the magnesium concentration for each template and primer combination. Mg²⁺ is an important player in PCR reaction. The recommended range of MgCl₂ to test is 2-4 mM; at lower Mg²⁺ concentration greater specificity.
• Use Hot Start DNA polymerase (like HotBegan™ Hot Start Taq DNA Polymerase).

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Yes. pSpark® TA and pSpark® TA Done vector could be used for cloning PCR products amplified with blend enzymes without any additional step, because such amplified DNA is a mix of both 3 ́-Adenine overhanging and blunt ended DNA molecules.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

• The restriction enzyme did not cleave efficiently: we suggest to cleaving near the end of a PCR fragment leave at least 4-6 bases past the restriction site, although it depends on enzyme. Test the restriction enzyme on a control substrate.
• The restriction enzyme was not completely inactivated: Phenol/EtOH purify the DNA or Clean-up if the enzyme cannot be heat inactivated.
• Star activity from the restriction digest cleaved the vector or insert. Check the DNA on a gel. If there is an extra band, reduce the amount of enzyme or time for the digestion.
• The DNA or restriction enzyme is contained exonuclease or phosphatase that damaged the ends.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

You can try 5-10% DMSO, up to 10% glycerol, 1-2% formamide, up 3x G-C Enhancer or combinations thereof. These compounds will lower the optimal annealing temperatures of your primers in 5-6 ºC.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

• You must purify the Ligation reaction prior to electroporation. The Buffer and the T4 DNA Ligase must be removed to prevent arcing of E.coli cells. Try phenol extraction or DNA clean up columns for remove this buffer and T4 DNA Ligase.
• You must use about 10 µg tRNA or glycogen carrier for DNA transformation.
• The electroporation should be optimized for the volume of the mix DNA and competent cells (like CVX5α™ Chemically Competent Cells).

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Up 10 % DMSO is used in PCR or cycle sequencing reactions of GC rich DNA in the presence of 2x the amount of Horse-Power™ Taq DNA Polymerase.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Horse-Power™ Taq DNA Polymerase is active from pH 7.5- 9.5.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

PCR does not requires a highly purified DNA template. However, it is very important that the DNA sample does not contain traces of some substances which are used in purification protocols and strongly inhibit the DNA Polymerase. PCR reaction can affect their performance if in DNA template solution remains Salts, Phenol, Etanol, EDTA, Proteinase K, Haemoglobin, etc.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

pSpark® DNA Cloning Kits includes cloning vectors for both Blunt-end fragments as PCR fragments amplified with non-proofreading DNA Polymerases. If you are using any proofreading (High Fidelity) DNA polymerase (like FastPANGEA™ High Fidelity DNA Polymerase), you should used pSpark® I, II, III, IV, V and pSpark® Done. But if you employ Taq DNA polymerase (like Horse-Power™ Taq DNA Polymerase) or any other non-proofreading DNA polymerase you must clone amplified fragments into pSpark®-T vectors: pSpark®-TA or pSpark®-TA Done.

PCR product from blend polymerases  is an heterogenous mixture of blunt end fragments (aprox 30%) and 3´-adenine overhangs fragments (aprox 70%). So the product of such PCR can be cloned into any vector of pSpark® family.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Yes. pSpark® DNA Cloning systems can be used for cloning PCR products amplified with enzyme blends without any additional step such as a blunting step. In fact, when such inserts were cloned in parallel into a T/A based vector and into pSpark® DNA Cloning Kits, a slightly higher number of colonies were obtained with pSpark® DNA Cloning systems using 10 times less DNA. No additional blunting step is needed because such amplified DNA is a mix of both 3´-Adenine overhanging and blunt-ended DNA molecules.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Taq DNA polymerase (like Horse-Power™ Taq DNA Polymerase) amplified PCR products can be cloned directly into pSpark® TA or pSpark® TA Done. It is not necessary any additional step.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

No, there is no need to purchase phosphorylated primers nor to phosphorylate the PCR product. Any primer you already have in your lab can be used for cloning into pSpark® DNA Cloning Kits. Phosphorylated primers do not inhibit ligation and thus can also be used. There is no need to add any specific sequence at 5´-ends of primers for cloning.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

All pSpark® DNA cloning Kits have Ampicillin resistance for selection of transformants and one vector has dual Ampicillin/Kanamycin antibiotic resistance. All inserts can be amplified by PCR with pUC/M13 forward and reverse primers and in vitro transcribed with either T7 or SP6 RNA polymerases. Blue white screening is possible in all vectors except pSpark® DNA cloning systems IV and V that are transcription free.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

No. Just set up a ligation according to its Product Manual, incubate ligation up to 60 minutes at 22 °C (or overnight if needed) and follow all the regular protocols you are familiar with. Use a 3:1 to 5:1 (recommended) insert-to-vector molar ratio as starting point. It corresponds exactly to 30 ng of insert per kb for the recommended 5:1 insert-to-vector ratio. As a rule of thumb you can use 30 ng per kb of insert to be cloned, that is, 15 ng, 30 ng or 60 ng if cloning an insert of 0.5 kb, 1 kb or 2 kb. If cloning an unpurified PCR product use 1 µL of PCR directly for ligation.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

pSpark® DNA Cloning based systems are about 50 times more efficient than any T/A based Cloning kits. As a result you will need about 10 times less DNA to obtain 4-5 times more colonies with a very low background (about 1%) and a very low percentage of cloning reaction failures and artefacts.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

pSpark® DNA Cloning Kits are  the most efficient cloning kits developed to date and in fact this supplied fast transformation protocol has been developed and tested to be used with it. Using this fast protocol of only 5 minutes transformation, cells with a transformation efficiency of 4×10⁷ cfu/µg and the supplied Control Insert, about 1,000 positive white colonies are obtained.

If you feel uncomfortable with this protocol, use the standard protocol, but as a suggestion to save your precious time, we recommend to test this protocol. We do not know, however, if for example, the method to prepare Competent Cells, the E. coli strain used, the temperature inside air flow cabinet, the transformation protocol used or the plating method, have influence in outcome. To date we have no customer who have tested this fast transformation protocol for cloning into pSpark® with unsatisfactory results.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

Yes, but we hihgly recommend to use the Canvax´s highly active Ligase with the lowest nuclease activity  T4 DNA Ligase included in the kit.  If you use another T4 DNA ligase please check both the total number of white colonies and the ratio of white positive colonies to blue negative colonies using the supplied Control Insert. This ratio is about 100 for the supplied T4 DNA Ligase. If you obtain less than 1,000 white colonies (using cells with 4×10⁷ cfu/µg) and a ratio of less than 20 then maybe our T4 DNA Ligase can help you in your laboratory.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

No. The vector vial has only a highly stable DNA vector and this contributes to the very high stability of pSpark® DNA Cloning vectors that could be even shipped at Room Temperature (only samples and kits without T4 DNA Ligase). We have stored freeze-dried pSpark® DNA Cloning Kits up to 30 days at 20-24 °C with no loss in cloning efficiency. However, we recommend the storage of vector at -20 °C.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.

pSpark® DNA Cloning Kits has been developed as an efficient system to clone blunt PCR fragments generated with High Fidelity Polymerases (like FastPANGEA™ High Fidelity DNA Polymerase). Currently pSpark® vector family also includes T-vectors for cloning PCR products obtained with Taq DNA Polymerases (like Horse-Power™ Taq DNA Polymerase), blend formulations or related Polymerases that add an adenine no template at the 3′ ends.

If you would answer more Questions, visit our FAQs or Expert Tips & Tricks Section.