Gateway Recombination and Seamless Cloning Support – Getting Started
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After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see Pg 17 of the Gateway® Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the Purelink® Quick Gel Extraction kit.
There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO® vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.
The smallest insert that has been successfully cloned into a pDONR vector in-house is 70 bp and the largest is 12 kb. Although successful cloning of small inserts from 50-200 bp is sequence-dependent, literature suggests that very small inserts (~30 bp) will not clone efficiently (Cheo et al., Genome Research, 14:2111-2120). For inserts larger than 70 bp, the efficiency of cloning decreases as the insert size increases. For inserts larger than 5 kb, we recommend an overnight incubation of the BP cloning reaction.
pDONR201 and pDONR207 can be used, but they replicate less efficiently than the Donor vectors we offer, pDONR221 and pDONR/Zeo even though both pDONR201 and pDONR207 contain the pUC ori. This results in lower plasmid yields. With pDONR221, plasmid yields are in the range of 0.5 - 1.0 µg of DNA per mL of culture.
The backbones of the pDONR221 and pDONR/Zeo vectors are exactly the same except for the antibiotic resistance marker. pDONR221 has the kanamycin antibiotic resistance marker, while pDONR/Zeo has the zeocin resistance marker.
For the most efficient BP reaction, it is best to not have attB sites in molar excess of attP sites. The standard BP Reaction (20 µL) uses 300 ng (no more than 500 ng) of pDONR vector and 30-300 ng attB-flanked PCR product or Expression Clone for 1 hour at 25 degrees C. Using too much of the donor vector in the reaction tube will inhibit the BP reaction and also result in intact donor vector being co-transformed with the Entry Clones. This will reduce the number of colonies on the plate by killing the transformed E. coli due to the presence of the ccdB gene. Longer incubation times of up to 24 h can be used to convert a higher percentage of starting attB-DNA to product. For PCR products > 4 kb, the number of colonies obtained per fmol of PCR DNA added decreases with increasing size. Thus, for larger PCR products, it is recommended to increase the amount of DNA to at least 100 fmol of PCR product per 20 µL reaction, and using incubations longer than one hour (e.g., 6 hours or overnight to 24 hours). The largest PCR-amplified DNA cloned in-house was 10.1 kb. Increasing the incubation to 4-6 h will typically increase colony output 2-3 fold and 16-24 h will typically increase colony output 5-10 fold.
You may continue but the recombination efficiency would drop by approximately 10 fold.
All Donor vectors and Destination vectors contain the ccdB cell death gene to reduce background of non-recombined BP/LR plasmids. Therefore, growing non-recombined vector requires special cells (One Shot® ccdB Survival 2 T1R Competent Cells) which are resistant to the lethal effects of ccdB. On the other hand, general E.coli cloning strains including TOP10 or DH5a may be used for plating the BP or LR reaction, or for propagation and maintenance of recombined Gateway® constructs.
Although sequential BP and LR reactions will provide you with the highest efficiency in terms of colony counts, our R&D scientists have developed a Single-step BP/LR Clonase™ reaction protocol which allows for simultaneous BP and LR reaction all in a single tube. We would recommend that this single-step BP/LR Clonase™ reaction be used for average-sized single inserts to save time. For large insert sizes or for Multi-Site Gateway™ cloning, we would recommend to stick with the standard two-step procedure.
Here are links to the publication of this Single-Step BP/LR Clonase™ reaction method and protocol:
BP Clonase® and LR Clonase® have the 5X BP reaction buffer or the 5X LR reaction buffer, respectively, in separate vials and require storage at -80 degrees C. On the other hand, BP Clonase® II and LR Clonase® II offer the convenience of pre-mixed BP Clonase®/LR Clonase® and 5X BP/LR reaction buffer, respectively. These pre-mixed formulations are more stable at -20 degrees C
LR Clonase® Plus has been discontinued and has been replaced by LR Clonase® II Plus. LR Clonase® II Plus is specially optimized for MultiSite Gateway® recombination reactions. LR Clonase® Plus used to have the enzyme and 5X LR buffer in separate vials and required storage at -80 degrees C. LR Clonase® II Plus offers the convenience of premixed LR Clonase® Plus and 5X LR reaction buffer in the same vial. This pre-mixed formulation is more stable at -20 degrees C. MultiSite Gateway® LR Clonase® II Plus provides the highest recombination efficiency for MultiSite recombination reactions. You may use LR Clonase® II Plus for single fragment recombination, but you cannot use standard LR Clonase® or LR Clonase® II for MultiSite recombination reactions.
No, this is not really feasible due to the fact that the attL sequences are approximately 100 bp in length, which is too long for efficient oligo synthesis and would also cause issues with the success of the PCR reaction. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.
No, since a stop codon would be necessary for an N-terminal tagged destination vector whereas the presence of a stop codon would block expression of the C-terminal tag.
The core region of attB and attL sites contains the recognition sequence for the restriction enzyme, BsrG1. Hence, we recommend using BsrGI for cutting out inserts from Gateway® vectors and validating successful BP and LR reactions. The recognition sequence for BsrG1 is TGTACA. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway® plasmids. Inserts can be released from all pENTR and pDONR vectors except pDONR P4/P1R that is part of the MultiSite Gateway® 3-Fragment System and that has an attL4 site whose sequence is different from attL1 and attL2. If a different restriction site is desired, the appropriate sequence should be incorporated into the insert by PCR.
We do not offer any plant Destination vectors but you could use the Gateway® Vector Conversion kit (Cat. No. 11828-029) to Gateway®-adapt plant expression vectors. We do offer pDONR P3-P4 and pDONR P5-P6 vectors as a custom service. 10 µg of each vector is supplied and they are only provided as a pair. If you are interested in this service, please send an e-mail to email@example.com
Donor vectors and Entry vectors contain two transcription termination sequences (rrnB T1 and T2) upstream from attP1 or attL1 or attR1 respectively. This prevents read-through transcription of genes cloned into these vectors, from other vector-encoded promoters thereby reducing possible toxic effects.
The pCR8/GW/TOPO® entry vector uses spectinomycin for selection so that the entry clone that is generated can be used with any Destination vector. Most Destination vectors have ampicillin resistance although there are a few that have kanamycin or zeocin resistance. Otherwise, the background will be too high, unless the entry vector is first linearized. Spectinomycin is a less commonly used selectable marker. We do not offer spectinomycin; spectinomycin dihydrochloride is available from Sigma, Cat. No. S4014
We previously recommended linearizing the Destination vector for more efficient recombination. However, further testing in-house has shown that linearization is not required to obtain optimal results. If the Destination vector is too large (>10 kb), linearizing it will help. We recommend linearizing the Destination vector by cleaving at a restriction site within the region of the Gateway® cassette, taking care to avoid the ccdB gene.
You would have to perform a BP reaction with a Donor vector, followed by an LR reaction with the new Destination vector.
No, the Shine-Dalgarno sequence does not adversely affect mammalian expression when used in an appropriate mammalian DEST vector.
The A, B, and C reading frame cassettes differ by 1 nucleotide each, allowing you to generate attR sites in all 3 reading frames. Each reading frame cassette contains a unique restriction site to allow you to distinguish between them (please see table on Page 3 of the manual).
There are a few things to check for:
- att sites must be appropriately labeled as a Miscellaneous Recombination feature type (as in attR1 or attR2);
- the ccdB CDS must be annotated on the vector (as “ccdB”) and
- the vector must be circular.
If the molecule is still not recognized as a Gateway® clone, compare the sequence of the att sites with the sequences of a similar att site which is recognized as a Gateway® vector. If there are a few point mutations, edit the att sequence on the vector which is not recognized to match a vector which is recognized. Proceed with the Gateway® cloning in the Vector NTI® program.
The single nucleotide differences are non-essential nucleotides in the att sites, but older versions of the Vector NTI® program have strict requirements to recognize Gateway® cloning vectors.
The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway® Destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing Donor vector such as pDONR™221.
The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your Destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.
There is no theoretical size limitation. PCR products between 100 bp and 11 kb have been readily cloned into a pDONR™ Gateway® vector. Other DNA pieces as large as 150 kb with att sites will successfully recombine with a Gateway®-compatible vector. Overnight incubation is recommended for large inserts.
We offer One Shot ccdB Survival™ 2 T1R cells (Cat. No. A10460) for propagating ccdB-containing plasmids.
The sequence of the control template is proprietary.
We do not offer any Gateway® vectors for expression in plants.
We do not offer the 5X LR Clonase® buffer and 5X BP Clonase® buffer as standalone products. They are available as part of the enzyme kits.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
Yes, we have come up with a single-step protocol for BP/LR Clonase® reaction, where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
In the single-step protocol for the BP/LR Clonase® reaction, we would not recommend substituting the BP Clonase® II/LR Clonase® II enzymes with BP Clonase® /LR Clonase® enzymes as this would result in very low recombination efficiency.
We recommend storing it at 4°C.
Mini-prep (alkaline lysis) DNA preparations work well in Gateway® cloning reactions. It is important to remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P.™ nucleic acid purification kits, ChargeSwitch® kits, or PureLink® kits is recommended.
MultiSite Gateway® Cloning
Please note that the MultiSite Gateway® Pro kits (2.0, 3.0, and 4.0) and the MultiSite Gateway® 3-fragment Vector Construction Kit have been discontinued as of December 31, 2015. The alternative product is the MultiSite Gateway® Pro Plus Kit (Cat. No. 12537100) for cloning of up to 4 DNA fragments.
With the MultiSite Gateway® Pro kits, you can use any Destination vector containing attR1 and attR2 sites whereas the MultiSite Gateway® 3-fragment Vector Construction Kit contains a specialized Destination vector with specialized att sites. On the other hand, the main advantage of the MultiSite Gateway® 3-fragment Vector Construction Kit is that you can use a standard entry clone with attL1 and attL2 sites. The enzymes are compatible between the two kits, but not the vectors.
Yes, we do carry pcDNA6.2/V5-PL-DEST that is promoter-less. You would use this vector if you wanted to clone in your own 5′ promoter using the MultiSite Gateway® Pro kit.
Sorry, we do not offer any of the MultiSite Gateway® vectors as standalone products. With the MultiSite Gateway® 3-fragment Vector Construction Kit (discontinued as of December 31, 2015), the pENTR 5′-TOPO® vector can be used to generate the entry clone containing the 5′ element (instead of doing a BP reaction with pDONRP4-P1R), and an entry vector with attL1 and attL2 sites can be used to generate the entry clone containing the gene of interest (instead of doing a BP reaction with pDONR221), but the entry clone containing the 3′ element would still have to be generated by doing a BP reaction with the pDONRP2R-P3 vector provided in the kit. With the MultiSite Gateway® Pro kits (2.0, 3.0, and 4.0 discontinued as of December 31, 2015; alternative is the MultiSite Gateway® Pro Plus Kit, Cat. No. 12537100, for cloning of up to 4 DNA fragments), however, the donor vectors provided in the kit have to be used to generate the required entry clones.
Standard MultiSite Gateway® reactions are incubated for 16-18 h at 25 degrees C. Two or 4 µL (for 4 fragment recombination) aliquots are transformed into One Shot® Mach1 cells. Use Mach1 cells instead of TOP 10 cells for the highest efficiency. For 2- and 3-fragment recombination reactions, it is important to keep optimum molar ratios of the plasmids (20 fmoles pDEST + 10 fmoles pENTR vectors). Hundreds of AmpR colonies can be obtained with a cloning efficiency of 90% (expected efficiency is a bit lower for 3-fragment recombination, usually 70-90%). For 2-fragment recombination dilute the reaction 1:10 in SOC medium and plate 50 and 100 ul. For 3-fragment, plate 50 and 100 ul of each transformation. For 4 fragment recombination use 10 fmoles of each pENTR plasmid plus 20 fmoles of the pDEST vector. Incubate for 16 hours and transform into Mach1 cells. The number of colonies and transformation efficiency is normally reduced for 4 fragment recombination. The number of correct clones has been observed to be as high as 80% and as low as 30%, depending on the inserts being cloned.
The MultiSite Gateway® Pro 2.0, 3.0, and 4.0 kits have been discontinued as of December 31, 2015. The alternative product is the MultiSite Gateway® Pro Plus Kit, Cat. No. 12537100, for cloning of up to 4 DNA fragments.
pDONR 221 is provided as a positive control for the BP recombination reaction, but cannot be used to generate multi-fragment entry clones in the MultiSite Gateway® Pro kit.
For the LR reaction, all Entry clones can be substituted with the supplied Control Entry clones. The number of colonies expected and/or the phenotype of the resulting clones are used to determine the efficiency of the LR recombination reaction. In the unlikely event that the attR sites in the destination vector are incorrect, then the LR reaction will result in zero clones. By performing one-by-one vector substitution reactions, the entry clone that is flawed can be identified.
- For the 2-Fragment LR Clonase® II Plus Positive Control recombination reaction, 2,000 – 15,000 colonies are expected if the entire 10 µL LR reaction is transformed
- For the 3-Fragment LR Clonase® II Plus Positive Control recombination reaction, 1,000 – 5,000 colonies are expected if the entire 10 µL LR reaction is transformed.
- For the 4-Fragment LR Clonase® II Plus Positive Control recombination reaction, 50 – 500 colonies are expected if the entire 10 µL LR reaction is transformed.
GeneArt® Seamless Cloning Kits
The GeneArt® Seamless PLUS kit is an improved version of the original kit. It is recommended for the assembly of up to 4 fragments and a vector totaling up to 40 kb compared to 13 kb for the original. The GeneArt® enzyme mix is also provided as a 2X mix with buffer that can be stored at -20 degrees C instead of -80 degrees C. Finally, the kit comes with the linearized pYES7L vector and Stbl3/pRK2013 cells that allows for horizontal transfer of the construct into a variety of recipient strains. Use this table to select the GeneArt® Seamless Cloning and Assembly kit that works best for your application.
The DH10B T1 SA cells are optimized for cloning large assemblies and we don’t recommend substitution with other competent cells.
We recommend at least 15 bp of homology for your GeneArt Seamless cloning and we have also tried 40 or 80 bp for larger inserts.
In theory, the GeneArt® Seamless cloning system can be used for library construction but we have not tested either application. Adapters with the required homology to the cloning vector would have to be generated.
We suspect that there would be some degree of preference for shorter fragments. We have seen 100% cloning efficiency with a 5 kb fragment, but the colony output was lower when compared to a 2 kb fragment. For example, you get about 400 colonies per 1 µL reaction for 5 kb and about 1200-2000 colonies per 1 µL for 2 kb. Also, we have observed in assemblies of larger fragments like 5 kb that if the PCR reaction of the 5 kb fragment is not gel-purified and there is a significant PCR band at a smaller size, then the smaller fragment tends to go in more than the 5 kb. We have not observed anything like this in fragments of 1 kb or 2 kb.
We don’t recommend over-incubating since the enzyme mix may chew back too much resulting in deletions. Shorter incubation times (e.g., 20 min) may be okay. For 4 fragments and 1 vector, we have tried 15 degrees C, RT and 30 degrees C and the best results were at RT with 77% cloning efficiency. The other temperatures gave us 31% and 37% efficiency. We do not recommend incubating on ice as you may get a lot of deletions at the junctions.
Colony numbers depend on a lot of factors, including how you prepare your vector, the vector ends (3′ overhangs give the highest colony output, 5′ overhangs give less, and blunt ends are close to 3′ overhangs), transformation efficiency, size of the inserts, and freshness of the inserts. An expected colony number range is as follows:
1 fragment cloning (1+1): 500-15,000 colonies/µL reaction
2 fragment (1+2): 500-10,000 colonies/µL reaction
3 fragment (1+3): 200-2,000 colonies/µL reaction
4 fragment (1+4): 50-2,000 colonies/µL reaction
We do not recommend this as cloning efficiency/colony output can decrease. Here are some tips to increase the likelihood of a larger assembly working:
- Make sure vector background is low – RE cut the vector, gel purify, then PCR amplify the vector. If PCR amplification is not possible, you can do a second cleanup to avoid inhibition after gel purification.
- Try a ratio of 1:1 instead of 1:2.
- Do not transform more than 6 µL, and do not use OmniMAX 2 cells even though they have a higher efficiency. TOP10 and MAX Efficiency DH5alpha work best.
- Try a longer recovery time (2 hours) after addition of SOC. Use 950 µL SOC, incubate for 2 hours at 37 degrees C, and then spin down the cells. Remove ~800µL and plate the rest on one plate.
- Longer overlaps (80 nt, for example) are better for large constructs. If the fragment ends have long overlaps, it may work better to try incubating for 45 min – 1 hour. However, small fragments (300 bp) may be negatively affected by this longer incubation – the enzyme will chew back the ends too much.
The smallest we’ve tested is 100 bp (>95% colonies contained insert). We haven’t tried annealed oligos for this.
There is no size limit on the individual fragments as long as the combined total length of the 4 individual fragments and vector does not exceed 13 kb.
There is no size limit on the individual fragments as long as the combined total length of the 4 individual fragments and vector does not exceed 40 kb.
This should not be a problem. An overlap that is a few bases shorter than recommended should still function in the reaction. However, for best results always use 15 bp.
GeneArt® High-Order Genetic Assembly System
We recommend having a total overlap of 50 nucleotides with adjacent fragments for constructs larger than 60 kb. For constructs smaller than 60 kb, a total overlap of 30 nucleotides is recommended.
We have developed the kit using desalted oligonucleotides, but higher purities may result in slightly better cloning efficiencies, especially during fragment editing.
Yes, the kit can assemble up to 5 fragments and 3 oligo stitches.
If the fragments are all below 5 kb and the total size of the molecule is below 13 kb, we recommend the GeneArt® Seamless Cloning and Assembly (Plus) kit. If you are assembling elements that have no end-homology, are too large to be amplified by PCR, or are trying to create a molecule over 13 kb, we recommend the GeneArt® High-Order Genetic Assembly System. Overall, the High-Order system can do more fragments, fragment editing, and oligonucleotide stitching and can do it at a higher efficiency. However, the assembly is in vivo (yeast) and takes longer to get the final construct due to longer growth periods for yeast compared to E. coli. Use this table to select the GeneArt® Seamless Cloning and Assembly kit that works best for your application.
The online web tool is not set up to do fragment editing automatically but it can be used to do it manually. The manual has a section on fragment editing that gives examples on how to use the tool for this purpose.
Use this table to determine the best cloning method to use for your application. For simple PCR cloning or subcloning of one gene smaller than 5 kb in a vector, our regular TA or TOPO® TA kits is the best option. Gateway® is the best expression platform there is for performing expression work in different hosts or moving genes from a vector to another routinely in their lab without the need to amplify the gene by PCR to clone it again. The GeneArt® cloning and assembly kits are for customers looking to clone more than 1 DNA fragment at once in a vector in a seamless way, those using large (over 5 kb) DNA fragments for which other cloning methods are less efficient, and for those that need seamless fusions without any extra sequences. The new platforms are also very flexible, and customers can use their own vectors to make DNA plasmids of up to 110 kb.
Cloning efficiencies can vary greatly based on different number of fragments with end-homology. Please find below approximate cloning efficiencies of assembled fragments into pYES1L:
- > 90% for 5 DNA fragments of 10 kb each
- > for 10 DNA fragments of 5 kb each
- > 50% for 10 DNA fragments of 10 kb each
For pre-existing fragments without end-homology assembled into pYES1L using ‘stitching’ DNA oligonucleotides, common cloning efficiencies are:
- > 90% for 1 fragment of 10 kb
- > 75% for 2 DNA fragments of 10 kb each
No, you can use enzymes that leave blunt ends or A-overhangs. Cloning efficiency will not be affected.
The pYES1L vector, a 9.4 kb linearized YAC-BAC shuttle plasmid, is a control vector for the assembly of DNA fragments in MaV203 cells and for the transfer of the assembled recombinant DNA molecule into E. coli. You can also use the pYES1L vector as a cloning vector, if desired. The origin of replication for yeast is Chromosome II’s centromere (single copy, high capacity YAC). The origin of replication for E. coli is F’ ori (single copy, high capacity BAC). Spectinomycin is used for selection in E. coli. Four reactions (1 tube with 8 µL) of the pYES1L vector are included with the kit. This is enough for control reactions, for cloning your inserts, or a combination of both. The pYES1L vector is also available separately (Cat. No. A13287, 10 reactions).
Large fragments (>5kb) are more susceptible to damage in a gel extraction procedure. Therefore, when using DNA fragments generated by PCR, we recommend that you assemble multiple fragments of less than 5 kb in one reaction rather than a single large fragment. Additionally, try to minimize UV exposure of the DNA and/or limit EtBr amount used. If you are attaching the insert to the linearized cloning vector, all nucleotides providing the requisite homology must be on the 5' end of the primer. If you are connecting two adjacent inserts, you may split the 30- to 50-bp homology between the fragments (e.g., 15 bp on the reverse primer of fragment 1 and 15 bp on the forward primer of fragment 2 for a 30-bp homology, or 25 bp on the reverse primer of fragment 1 and 25 bp on the forward primer of fragment 2 for a 50-bp homology). Please note, you can split the homology between adjacent fragments in any combination (e.g., 15+15 as in the example above or 20+10, 25+5 etc. for a 30-bp homology).
There is no size limit on the individual fragments as long as the combined total length of the 10 individual fragments and vector does not exceed 110 kb.
The Lysis buffer is only available as part of the kits and is not offered as a standalone item.
The CSM Media is available as a standalone item (Cat. No. A13292).
We recommend using our Platinum® PCR Supermix High Fidelity enzyme (Cat. No. 12532-016) for amplifying DNA fragments up to 5 kb for general assembly applications. If you require higher PCR fidelity, we would recommend one that has processive and proof-reading capabilities.
Stitching oligonucleotides used for insertion editing must have a 30-nucleotide overlap with each adjacent fragment in addition to the insertion bases (for a total length of up to 80-mer, including up to 20 insertion bases). See manual for diagram. Note: This applies for a 2-fragment assembly and the insertion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.
Stitching oligonucleotides used for deletion editing must have a 40-nucleotide overlap with each adjacent fragment, annealing up to 6 nucleotides from the junction into each fragment, thus leaving up to 6 bp at the end of each fragment to be deleted during transformation-associated recombination. See manual for diagram. Note: This applies for a 2-fragment assembly and the deletion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.
Yes, you should be able to adapt your E. coli vector into a yeast-compatible cloning vector using the GeneArt® High-Order Vector Conversion Cassette (Cat. No. A13291) for use with the GeneArt® High-Order Genetic Assembly System with the following provisions:
- Start by using the DNA Oligo Designer web tool, and verify that your vector and the GeneArt® High-Order Vector Conversion Cassette do not share internal homology to prevent potential re-arrangements when using your adapted vector with the GeneArt® High-Order Genetic Assembly System.
- Use a vector with a single- or low-copy-number origin for a final construct of >15 kb, if the final plasmid construct will be transferred into E. coli. Usually, low-copy-number E. coli vectors have significantly higher capacity than high-copy number vectors.
- Avoid chloramphenicol selection markers on the custom vector since this is the marker on the cassette.
- After ligation (1:10 vector: insert ratio recommended), transform competent E. coli cells with the ligation mixture and plate on double selection LB plates (chloramphenicol plus the antibiotic marker on your custom vector backbone). To linearize your yeast-adapted cloning vector for multi-fragment assembly, a double-digestion is required to avoid background caused by residual palindromic end sequences resulting from a single enzyme digestion.
The DNA Oligo Designer is a free online web tool that will help you to design your cloning construct and DNA fragments. It will help to eliminate potential pitfalls linked to your specific sequences, and performs in silico cloning using your sequences. It can also provide a graphic representation of the final assembled molecule as well as a downloadable GenBank file compatible with VectorNTI® software. Please note, this Designer does not support fragment editing. However, for deletion editing, you can enter the fragment sequence into the DNA Oligo Designer with the end deletions already applied. The designer is available here.
GeneArt® Type IIs Assembly
The GeneArt® Type IIs Assembly kits are based on “Golden Gate cloning” which relies on the unique properties of Type IIs restriction enzymes to generate compatible ends on DNA fragments that can then be joined together by T4 DNA ligase. These kits are designed for simultaneous and seamless assembly of up to 8 DNA fragments (ranging in size from 25 bp to 10 kb into a recipient vector for a total insert size of 10 kb plus the vector, totaling up to 13 kb) in a single-tube, through a restriction-ligation reaction. The assembly is not based on homologous recombination and hence minimizes the risk of rearrangements.
Each of the GeneArt® Type IIs Assembly kits supplies the GeneArt® Type IIs Enzyme mix (GeneArt® AarI, BsaI, or BbsI Enzyme Mix), pType IIs recipient vector and pType IIs–CTRL vector. For pre-cloning DNA inserts before assembly, we recommend using pCR™-Blunt II-TOPO® vector (Cat. No. 450245), as the pre-cloning donor vector. Do not use other TOPO® vectors or other vectors with additional AarI, BsaI, and BbsI recognition sites as pre-cloning vectors. To transform the assembled DNA construct, we recommend using chemically competent MAX Efficiency® DH10B™ (Cat. No. 18297-010) or electrocompetent ElectroMAX™ DH10B™ (Cat. No. 18290-015). For generation of DNA fragments by PCR, we recommend using AccuPrime™ Pfx SuperMix (Cat. No. 12344-040), because the AccuPrime™ Pfx DNA polymerase is ideal for high-fidelity, high-specificity amplification of DNA fragments. To purify the DNA inserts, we recommend that you use the PureLink® PCR Purification Kit (Cat. No. K3100-01) or the PureLink® Quick Gel Extraction kit (Cat. No. K2100-12).
- When assembling ≤5 fragments using recommended protocols, at least half of the resulting clones will contain correct inserts in correct order and orientation.
- When cloning 6 or more fragments, we recommend screening more colonies as the cloning efficiency may be slightly lower.
Here are some suggestions for primer design:
- Design your PCR primers such that each DNA fragment to be assembled is between 25 bp and 10 kb in length.
Note: Large inserts (>5 kb) are more susceptible to damage in a gel extraction procedure. Furthermore, many PCR enzymes are not processive enough to amplify inserts >5 kb. Therefore, we recommend that you assemble multiple inserts of ≤5 kb in one reaction rather than a single large insert.
- 5′ ends of each primer pair (forward and reverse) must contain a 6-nucleotide end-cushion segment followed by the restriction enzyme recognition sequence (6 to 7 nucleotides, depending on the enzyme).
- 3′ ends of each primer pair must have at least 12 nucleotides of template specific sequence that is unique.
- Primers must not contain internally complementary sequences to avoid hairpin formation. Similarly, there must be no complementary sequences between primer pairs.
- Best results are obtained using primers with a sequence-specific partial Tm of 50 – 68 degrees C.
We also have the GeneArt® Primer & Construct Design Tool for primer design.
All three GeneArt® Type IIs Assembly kits contain the pType IIs recipient vector and pType IIs–CTRL vector. The only difference between the three kits is the Type IIs Enzyme mix that is present in the kit. To maximize the applicability of the system, the GeneArt® Type IIs Assembly Kits are available in three different enzyme blends based on the non-palindromic recognition sites of varying lengths and the % GC content: AarI (7 bp, 71% GC), BbsI (6 bp, 50% GC), and BsaI (6 bp, 66% GC ). Each of the three blends contains all required enzymatic and non-enzymatic components and comes as a single 2X concentrated mix, thereby simplifying experimental design and minimizing pipetting. We recommend selecting the appropriate kit based on the sequence of the fragment (s) being assembled.
- We recommend using pre-cloned fragments for assemblies of more than 5 fragments, assemblies using fragments that are <250 bp or > 2kb to increase the cloning efficiency and colony output during final assembly.
- We recommend using pre-cloned fragments for the assembly of repetitive or homologous sequences of at least 80% identity. You can assemble up to 4 precloned DNA fragments containing repetitive/homologous sequences into a recipient vector for a total construct size of 2.4 kb plus the vector. The precloned fragments must be between 150 bp and 600 bp in size.
- For best results, we recommend using pCR™-Blunt II-TOPO® vector (Cat. No. 450245), as your pre-cloning donor vector. Do not use other TOPO® vectors or other vectors with additional AarI, BsaI, and BbsI recognition sites as pre-cloning vectors.
Here is a table that illustrates the advantage of using pre-cloned DNA fragments for assembly over directly using PCR fragments:
|Fragment type||Maximum no. of fragments||Fragment size||Total construct size|
|Pre-cloned||8 + vector||25 bp – 10 kb||10 kb + vector|
|PCR||5 + vector||250 bp – 2 kb||10 kb + vector|
|Pre-cloned, with rep/hom*||4 + vector||150 bp – 600 bp||2.4 kb + vector|
|PCR, with rep/hom*||2 + 2 + vector**||20 bp – 500 bp||2 kb + vector|
*Fragments with repetitive/homologous sequences of at least 80% identity
If desired, you may retrofit your own vector to use as a recipient vector. The retrofitted vector must contain ccdB as a counter-selectable marker, flanked by at least one pair of identical recognition sites for the AarI, BsaI, and BbsI endonucleases. If more than one type of Type IIs recognition site is present in the vector, they must be included in symmetrically arranged MCSs separated by the ccdB counter-selectable marker. The vector must not contain any other copy of the Type IIs recognition sites outside of these MCSs.
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For Research Use Only. Not for use in diagnostic procedures.