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All products sold by ZyCloning LLC or Distributors are for end use only. There is no restriction on the end usage type. All resale in the original format, repackage, or part of the intended reagent without ZyCloning's license, are prohibited.

ZyCloning Master Mix (3x)

ZyCloning Master Mix (3x) list

ZyCloning Master Mix (3x) protocol and usage guide

As the major component of the ZyCloning system, the ZyCloning Master Mix (3X) is a user-friendly way to quickly clone gene fragments into target vectors successfully for transformation into appropriate complement cells.

Each product includes the ZyCloning Master Mix (3x) and 5 reactions of positive control (ZYPC1: ZY Positive Control).

Assembly Reaction Composition

Component                                                                        Volume

DNA Fragment 1 (vector) (3ng/kb)                                     1 μl

DNA Fragment 2 (or inserts) (9ng/kb total)                        1 μl

ZyCloning Master Mix (3X)                                                 1 μl

Total volume                                                                      3 μl

·   Positive control is a mixture of vector and insert, therefore, 2 μl of positive control and 1 μl of ZyCloning Master Mix (3x) is used for control experiment. The assembled product from positive control is a Kanamycin resistant plasmid with pMB1 origin and constantly express fuGFP, it makes the colony to be green under long wave UV light.

Assembly Protocol

Total reaction mix is Incubate at 72oC for 30 seconds then cooled on ice or at 4oC on a thermal cycler, after which the total reaction mix is ready for transformation into any competent cells suitable for cloning. The chemical competent cell from ZyCloning LLC, from preparation methods of TSS or CaCl2 method, can be used as rapid transformation procedure, there is no need for recovery after heat shock. The procedure can be performed on any quick heat instrument such as PCR thermocycler, heat block, water bath etc.

Application Notes

·           Homology base count references the 5’ end. The mismatch after homology region is removed either it is 5’ or 3’. The tested length to be removed is 14 bp count to 5’ end. The general rule is that mismatch is no more than 1/3 of the homology length.

·         The vector amount and insert amount should be in the designated range. If fixed molar ratio of vector: insert is 1: 3, The good range of vector amount is 3 to 6 ng/kb with acceptable amount is 1.5 to 12 ng/kb. If the vector is fixed at 3 ng/kb, the good amount of insert is 4.5 ng/kb to 18 ng/kb, with acceptable range is 2.3 ng/kb to 36 ng/kb. If in other cases, the total amount of all DNA fragments good range is 0.07 to 0.14 pmol, with acceptable range is 0.03 to 0.23 pmol. 

·       When in 3 pieces assembly, the vector: insert1: insert2 is recommended to be 1: 1.5: 1.5.  The ZY Cloning System is not suitable to assemble over 3 pieces. Instead, overlapping PCR is recommended for multiple fragments to be linked as one piece, then the PCR product is purified and processed as single piece. The correct assemble efficiency depends on the actual complexity.

·         The vectors from restriction enzyme digestion have higher efficiency than from PCR. The extra fragments from restriction enzyme digestion can be kept in the reaction as long as the fragments are absent of homologous region on either end.

·       Vectors with 5’ protruding end have higher efficiency than blunt or 3’ protruding ends. If the insert is 5’ protruding or blunt, it is fine for the insert to assemble with any type of ends vector. If the insert is from PCR with Taq DNA polymerase, it is observed that only the vector with 5’ protruding ends can be used for the assemble.

·         Synthesized gene fragments are appropriate for direct use. Purified PCR product increases the colony number. In this situation, it is better to add the ZyCloning Master Mix at the last step.

.        Crude PCR product can be used in the following conditions: the crude PCR is the insert with proof reading polymerases. Mix 2 ul of PCR product with 0.5 ul SAP and 2.5 ul 1mM Tris-HCl, 1 mM MgCl2  pH8.0, and incubate a room temperature for 20 min. There is no need to heat kill the SAP.  1ul of the treated PCR product is then mix with 1 ul prepared vector and 1 ul ZyCloning Master Mix for the assembly reaction. 

·         Each homology arm should be 12- 65 bps in length. And when short homology arm is used, make sure the Tm is at least 36 oC.

·         The shortest direct insert length is tested successfully is 131 bp.  To incorporate even shorter oligos, ensure they are synthesized with 5’-protruding 12-65 bp homology. The following assembly reaction is then recommended:  mix 2 μl of vector and 1 μl Zycloning Master Mix (3x) first, then add 1 μl oligos at 36 oC for 30 seconds, then cool down to the transformation procedure.

·           If the insert is a synthesized gene fragment, it should be free of any additional adaptors. Only if the adaptor is shorter than 1/3 of the homology region, then it can be ignored.

·           For 2 pieces inserts, the homology region is recommended to be 24 bp.

·          Using crude primers in PCR potentially reduces homology length; therefore, it is recommended to add 6 additional bases to crude primers.

·   The thin walled 0.3 ml PCR tube is recommended for the assemble reaction. For reactions in vessels with thicker plastic walls, the reaction time needs to be elongated.

ZyCloning Ready to Use and Whole Vectors

ZyCloning Ready vectors are verified ZyCloning vectors with 95% minimum insert rate with the control insert DNA. All ZyCloning Ready to use vectors are Kanamycin resistant with pMB1 origin. Homologous end requirements for insert are listed.  5 reactions of insert with appropriate sequence homology are supplied as positive control. All the ZyCloning Ready to use vectors are fully with 5’ phosphate.  While the cloning efficiency is high for the appropriate insert, the actual expression level and solubility of different proteins are still in variation.

ZyCloning whole vectors are the plasmids which can be converted into Ready to Use version by restriction enzyme BsaI and purification.

The vectors in the same group share the same homology region for seamless cloning.

7th Group Vectors

Features include a TEV site; for polypeptides initiated with M/G/S/C/A/H, the target protein is cleaved without additional amino acids. Protein expression is comparable to 5th Group Vectors. TEV digestion relies upon the initial amino acid of the expressed polypeptide being S or G for highest efficiency, but general starting M is a good target also.

Required insert homology for gene fragment synthesis:

5’  CTGTACTTCCAG

3’  TGATAGTCGGCT

For PCR, ensure that the reverse primer sequence is reverse complementary. 

8th Group Vectors

8th Group Vectors are the ones with tags on the C-terminal. The initial design is the linker plus tags. The C-terminal protease site between target protein and tags are optional and to be included in the gene if needed. If a stop codon is placed before the tag, native protein can be expressed.

Required insert homology for gene fragment synthesis:

5’  GGAGGTAAATAAATG

3’  GGTTCTGGAAGT

For PCR, ensure that the reverse primer sequence is reverse complementary. 

ZyCloning Utility plasmids

ZyCloning Restriction Endonuclease

DpnI is the DNA restriction enzyme to remove Dam methylate template plasmid in many site-directed mutagenesis.

However, there are chances the digestion fails, either from the enzyme itself or buffer condition and digest time.

In theory, very little amount of template

 DNA can reduce the final colonies containing wild type, but in reality,  PCR can fail if the template is too little.

By adding same amount of GFP expression plasmid after the mutagenesis PCR but before the adding of DpnI enzyme, the green colonies can be an indicator of a failed DpnI digestion. It is way better than the results is only verified by culture grow, plasmid extraction then sequencing.

It only approve the activity of DpnI, since template in PCR can go one round of PCR to form hemi dam methylated DNA which is much harder to digest, the colonies without fluorescent under UV light are not guaranteed to be right mutant, sequencing is still needed for final verification.

ZyCloning LLC provides critical restriction endonuclease for the molecular cloning purposes.

Sor far the only restriction enzyme provided in the purified enzyme form, is the BsaI-ZY.

ZYR01: BsaI-ZY 5000 units, 100 units/ul, $100.00

10x Reaction buffer is provided with the enzyme.

ZyCloning Competent Cells

                 ZyCloning Competent cells are suitable for using a rapid transformation procedure.

This rapid procedure is designed for one or two inserts assembly. For drug resistance of ampicillin, kanamycin or chloramphenicol, the incubation after heat shock can be omitted before plating.  Antibiotic concentration for plates is recommended to be 40% of traditional antibiotic concentrations unless it is ampicillin alike, which is 100% as normal; however, the antibiotic concentration in culture media for colony growth should be equal to traditional concentration recommendations.

                 ZyCloning Transformation Enhancer is a reagent which can greatly enhance the transformation efficiency of ZyCloning Competent cells. For rapid transformation enhancement, 20 ul of enhancer is mixed with 50 ul ZyCloning competent cell, then the total 70 ul of mixture is added to the assembly product, after the 42 oC 30 seconds heat shock, whole 73 ul is plated on the LB agar plate with appropriate antibiotic. The rapid transformation efficiency can be enhanced around 4-fold. For normal transformation enhancement, 20 ul of enhancer and 14 ul of sterile water is mixed with 50 ul ZyCloning competent cell, then the total 84 ul of mixture is added to the assembly product. After the 42 oC 30 seconds heat shock, 450 ul of LB media is added and incubated at 37 oC and vigorously shake for one hour. Then the whole transformation mixture is spun down, all but 50 ul supernatant is discarded. The total cell precipitate is resuspended in the leftover supernatant, then the whole amount is plated on the LB plate with appropriate antibiotic. The normal transformation efficiency can be enhanced around 12-fold.

                 Note:  The chemical competent cell from other company mostly will work for ZY Cloning system if following their original transformation procedure. Most of them may not work in the rapid transformation procedure as described above. Electro competent cells are not compatible with ZY cloning system.

           The competency of these competent cells is subcloning efficiency if judged on traditional transformation method, is 1-2x105 colonies/ ug of pUC19. However, it produces hundreds of colonies on a ZY cloning positive control reaction mix in the procedure of rapid transformation.

DH10B genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara,leu)7697 araD139 galU galK nupG rpsL λ-

DH10BC genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara,leu)7697 araD139 galU galK nupG rpsL λ- pMC-RecA (pACYC origin, Camr)

DH10BT7 genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara,leu)7697 araD139 galU galK nupG rpsL λ- pBAD33-RecA-T7RNA Pol (pACYC origin, Camr)

In Rapid Transformation, the concentration of antibiotic is critical. Except ampicillin group antibiotics, other antibiotics require lower concentration as Kanamycin (20 ug/ml) and Chloramphenicol (13 ug/ml). The liquid culture for the colonies have the normal concentration. Tiny colonies may occur in the incubation, they won't grow up in the liquid culture with normal antibiotic concentration. The plate is at the size of 90 mm x 15 mm.

Production Strains

Exclusive production strain from ZyCloning, LLC, price at $10,000.00 plus 10% sale in the patent span

BsaI-ZY, DNA restriction endonuclease cut at GGTCTC(N1/N5), even less star activity than BsaI-HFv2 from New England Biolabs;

Nt.BstNBI-TL, DNA nicking endonuclease, nick at GAGTCN4^

Non Exclusive production strain from ZyCloning, LLC, price at $5,000.00

H5-BsaI, DNA restriction endonuclease cut at GGTCTC(N1/N5), N-terminal His tagged.

H5-Nt.BstNBI, DNA nicking endonuclease, nick at GAGTCN4^, N-terminal His tagged.

H5-DpnI, DpnI restriction endonuclease, cut at N6 methylated GAm6/TC

HX5-Nt.BspQI, restriction endonuclease, nick at GCTCTTCN1^, N-terminal is 6xHis and NEXT tagged.

To order instruction

To order: 

Send product list to zhuz@zycloning.llc for quote to start purchase; 

Tech support: zhuz@zycloning.llc

                               857-204-8289