What is the maximum length of gBlocks® Gene Fragments for robust Gibson Assembly® reactions?
The maximum length of gBlocks Gene Fragments is 500 bp. For fragments up to 500 bp, efficiency is not substantially affected by the length of the fragments.
The original paper describing the Gibson Assembly Method* suggests the method can be used to assemble constructs in the size range of small bacterial genomes, and megabase assemblies have been demonstrated using the Gibson Assembly Method.
*Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods 6 (5): 343–345. DOI:10.1038/nmeth.1318. PMID 19363495.
Your recommended overlap is 20–30 bp; the original paper recommended ~40 bp. Do you see any difference between overlap length? Is there is an optimal overlap length?
For short constructs-those smaller than 1.5 kb, we do not see differences in the overlap length used; however, we have not exhaustively tested the use of 20 bp overlaps. For ligating fragments to generate longer constructs, the use of overlaps up to 60 bp increases efficiency.
The original paper assembled oligonucleotides. Our recommendation is based our experience assembling longer gene fragments. In addition, part of the recommended length for the overlaps is to overcome specificity issues.
Can I use gBlocks® Gene Fragments to clone genes with a CAG repeat like those in the Huntington’s gene?
Individuals suffering from severe forms of Huntington’s disease often have 40 or more repeats of the CAG motif in the HTT gene. The large number of repeats would be extremely hard for us to sequence verify due to the enzymatic slippage that occurs by the sequencing polymerase in these motifs. In general, trinucleotide repeats larger than 3 will cause a problem for synthesis of gBlocks™ Gene Fragments.
What is meant by “estimated purity” with gBlocks® Gene Fragments?
We estimate purity of gBlocks Gene Fragments indirectly by sequencing cloned, transformed, and purified products resulting from isothermal assembly reactions and traditional ligation reactions. Because there are multiple sources of error in the measurement, including but not limited to the gBlocks Gene Fragments themselves, we can only estimate the purity of gBlocks Gene Fragments. Because we see similar errors when screening clones of DNA amplified directly from genomic DNA or fragments of DNA cut from previously sequenced plasmids, we believe the actual purity of gBlocks Gene Fragments is higher than what we observe after transformation.
Can you use gBlocks® Gene Fragments to change a few restriction sites in a plasmid vector?
Because gBlocks Gene Fragments can be synthesized up to 500 bp in size, they can be designed to replace a segment of a vector up to around 440–460 bp in size using the isothermal assembly method. This would make possible replacement of many restriction sites.
Following assembly, can the assembled gBlocks® Gene Fragments be cloned into a TOPO vector?
Isothermal assembly uses a 5’-3’ exonuclease (ExoV). When the assembly is done linearly (rather than cloning directly into a vector) the 5’ terminal ends are digested leaving single-stranded ends that will not be filled in. An additional step of PCR amplification, with forward and reverse primers, would be needed to create either blunt ends or ends compatible with T/A cloning, depending on which TOPO kit is to be used.
Do you recommend any software for designing the overlap regions in gBlocks® Gene Fragments for optimal Gibson Assembly® Method results?
Unfortunately, we do not have automated software developed for designing gBlocks Gene Fragments. However, our synthetic biology specialists at firstname.lastname@example.org can assist in designing such overlaps.
Is the Gibson Assembly® method as efficient for 500 bp fragments as with smaller fragments?
For fragments 200–500 bp, efficiency is not substantially affected by the length of the fragments. However, for fragments smaller than 200 bp, it is sometimes beneficial to add a higher ratio of fragments to vector, particularly if the fragments are AT rich.
Is there a minimal size of gBlocks® Gene Fragments that can be assembled with the Gibson Assembly® Method?
For manufacturing reasons, we offer gBlocks Gene Fragments of 125–500 bp in length. We have not tested assembly with shorter fragments, but we know from publications that it works on 60mer oligos.
Do we receive the Sanger sequencing traces when we get the gBlocks® Gene Fragments?
Unfortunately, we do not have a way to export the Sanger sequencing data from our QC, as the sequencing data contains additional, proprietary information. Upon request we do offer certificates of conformance that are signed by the technician who reviews the sequences.
What are the potential causes for errors in gBlocks® Gene Fragments? Is it the Gibson Assembly® Method or does it come from the gBlocks fragments themselves? What are the most common failures in the fragments generated via gBlocks synthesis?
gBlocks Gene Fragments contain a small population of incorrect fragments. These may be caused by erroneous bases in the Ultramer® Oligos used to make them, misassembly, and errors occurring during the amplifications. These errors are rare and usually consist of single-base deletions or substitutions, often near the ends of the gBlocks Gene Fragments.
Errors in the gene assembly and cloning process can result in large deletions, small deletions, and single-base changes.
Is there a place one can check a sequence intended for submission to verify that it meets IDT sequencing conditions?
Yes. Please contact email@example.com and one of our synthetic biology specialists can help with any design or sequence questions you may have.
Can you combine gBlocks® Gene Fragments with PCR fragments and digest fragments in one Gibson Assembly™ reaction (staying within 5 fragments of course)?
Yes. Isothermal assembly will work with PCR fragments, gBlocks fragments, and fragments digested using restriction endonucleases. Make sure to use a polymerase that does not add “A’s” at the end of the amplicon.
When using Gibson Assembly with plasmid-derived restriction fragments, do you need to purify the fragments by gel electrophoresis, or simply mix the crude digests?
Gel purification is not always necessary for efficient assembly. However, gel purification can help reduce background, particularly if the destination vector has the same antibiotic resistance as the plasmid-derived fragments.
Are gBlocks® Gene Fragments cheaper than MiniGenes™ Synthetic Genes?
Yes, gBlocks Gene Fragments are cheaper than MiniGenes Synthetic Genes. However gBlocks Gene Fragments do require assembly into the final construct (done in one step, that includes vector insertion), while MiniGenes Synthetic Genes are provided in a vector, ready to use.
We also recommend sequencing the resulting clones prior to gene expression. When cloning a single gBlock Gene Fragment, >80% of clones should contain the correct sequence.