gBlocks® Gene Fragments Frequently Asked Questions

  • Can gBlocks® Gene Fragments be used for microinjection as single guide RNA (sgRNA) expression cassettes for CRISPR applications?

    A recent report compared the efficiency of microinjection of DNA versus RNA in mouse embryos [1]. While it was shown that DNA is effective, in vitro transcribed RNA was observed to be more efficient.

    Typically, microinjections for CRISPR applications are performed using in vitro transcribed Cas9 and sgRNA rather than native dsDNA. gBlocks® Gene Fragments are ideal for use as template for in vitro transcription and will work well in these applications [2,3]. However, long RNAs are known to trigger the innate immune response in many cells, which increases the expression of dozens of genes and can affect cell viability and general health [4–7].

    References

    1. Horii T, Arai Y, et al. (2014) Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep, 4:4513.
    2. Niu Y, Shen B, et al. (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 156(4):836–843.
    3. Wang H, Yang H, et al. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4):910–918.
    4. Gantier MP and Williams BR (2007) The response of mammalian cells to double-stranded RNA. Cytokine Growth Factor Rev, 18:363–371.
    5. Judge A and MacLachlan I (2008) Overcoming the innate immune response to small interfering RNA. Hum Gene Ther, 19:111–124.
    6. Hornung V, Hartmann R, et al. (2014) OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids. Nat Rev Immunol, 14:521–528.
    7. Robbins M, Judge A, MacLachlan I (2009) siRNA and innate immunity. Oligonucleotides, 19:89–102.
  • Can you use gBlocks® Gene Fragments to alter restriction sites in a plasmid vector?

    Because each gBlocks® Gene Fragment can be 125−2000 bp in length, the fragments can be designed to replace a segment of vector up to 2 kb in size (allowing for 20–30 bp overlapping regions between the gene fragment and plasmid insertion site) using the Gibson Assembly® Method. This would make possible the replacement of many restriction sites, as well as adding tags and other useful sequences. Learn more about gBlocks Gene Fragments.

  • Should I amplify my gBlocks® Gene Fragments when I receive them?

    gBlocks® Gene Fragments are chemically synthesized, double-stranded DNA, delivered normalized to 250, 500, or 1000 ng, depending on length, and dried down. This should provide sufficient DNA for 2–4 cloning reactions according to most protocols. We recommend avoiding amplification of gBlocks Gene Fragments because of the potential for polymerase introduced errors. If you do need to amplify your gBlocks Gene Fragments, it is strongly recommended that you use a high-fidelity polymerase in order to limit the introduction of errors to your gBlocks Gene Fragments sequence. A protocol for amplification using the Phusion® DNA polymerase (NEB) can be downloaded at www.idtdna.com/gBlocks, under the Support tab. For additional information, download a copy of the gBlocks Gene Fragments user guide or contact us at genes@idtdna.com.
  • What quality checks do gBlocks Gene Fragments with variable bases receive?

    The constant regions are gBlocks® Gene Fragments and undergo size verification by capillary electrophoresis and sequence verification by mass spectrometry. The variable regions cannot currently receive complete analysis (using NGS, which would be needed for this analysis, would be cost prohibitive). We rely on a validated process that we have experimentally confirmed by NGS that ensures >80% of DNA species are present in the final material shipped.
  • What does sequence-verified mean for gBlocks Gene Fragments?

    gBlocks® Gene Fragments are double-stranded DNA fragments that are ideal for use in synthetic biology, gene construction and traditional cloning applications. They are manufactured using the same industry-leading, high-fidelity synthesis chemistries developed by IDT for our Ultramer® oligonucleotides, and then sequence-verified. However, as they are not cloned, a marginal number of molecules with small sequence errors remain. The incidence of these errors is no higher than that observed from PCR-amplifying an insert from a cloned plasmid and at least 85% of cloned gBlocks Gene Fragments will be of the correct sequence. As for every cloning event, IDT recommends verifying the resulting construct. Learn more at www.idtdna.com/gblocks.
  • Should I order my gBlocks Gene Fragment with the 5′ phosphate modification?

    Adding the 5′ phosphate modification to gBlocks Gene Fragments is unnecessary for most applications. The 5′ phosphate is required for traditional blunt-end cloning. Do not add 5′ phosphate for Zero Blunt TOPO® cloning (check manufacturer information).
  • How do I design my gBlocks Gene Fragment for restriction cloning?

    Cloning of gBlocks Gene Fragments is similar to cloning a very pure PCR product. For restriction cloning it is important to add 6–8 nt at the ends of the fragment, beyond the restriction recognition sequence. Most restriction enzymes will not cut efficiently, or at all, if the fragment terminates precisely at the restriction site.
  • What if I need a gBlocks Gene Fragment library that is more complex than what you offer online?


    We realize that this first offering for gBlocks Gene Fragments libraries is somewhat limited. We are working hard at offering more complex libraries in the future. Help us prioritize by sharing what you need from us in as much detail as possible (i.e., show us sequences including mixed bases, annotations, drawings, etc.) and send to libraries@idtdna.com.
  • Why am I limited to 18 N or K mixed bases in my gBlocks Gene Fragment?

    Incorporating 6 NNK codons corresponds to about 1 billion possible combinations, and 18 N mixed bases will create a pool with 68.7 billion different gene fragments. As the number of variable bases increases, the number of molecules representing a particular sequence decreases. We decided to limit variable regions to 18 mixed bases to give customers the best overall pool of library constructs. Since most functional screens cover 1,000–100,000 recombinant colonies, allowing 18 mixed bases should be adequate for producing meaningful results.

  • What are the N and K mixed bases that can be incorporated into the variable regions of gBlocks Gene Fragments?

    Amino acids are represented by 1–4 codons that are defined by the genetic code. NNK codons are commonly used in screens for codon substitutions to reduce the presence of some codon-rich amino acids, thereby reducing their overrepresentation in a particular library. Additionally, using NNK codons removes 2 out of 3 possible stop codons, which also limits the number of sequences in a library that produce unwanted truncated gene products.
  • Can I order multiple variable regions in my gBlocks Gene Fragment?

    Currently, only consecutive bases are allowed. Specifically, up to 18 consecutive N or K bases can be ordered, with a minimum of 125 bp of fixed, flanking sequence on either side of the variable bases. The maximum length for the entire sequence is currently 500 bp, including the variable bases.
  • Are N and K the only mixed bases are allowed for gBlocks Gene Fragments?

    Only N and K are available at this time. We are working on adding other mixed bases such as – R, Y, M, S, W, H, B, V, D. By letting us know what you need for your research on libraries@idtdna.com, you can influence how we prioritize our development efforts. In many cases, we can already offer suitable solutions, that are not offered online.
  • What is the length of synthetic/custom genes that IDT can synthesize?

    gBlocks™ Gene Fragments—sequence verified double-stranded DNA—are available from 125 to 2000 bp, MiniGene™ Synthetic Genes are available from 25 to 500 bp, and Genes, start at 501 bp. MiniGene and Genes products are supplied cloned into IDT vectors. For additional information on synthetic biology products, email genes@idtdna.com.

  • What types of sequence motifs should be avoided when ordering gBlocks® Gene Fragments?

    Sequences that cause particular problems for gBlocks Gene Fragment synthesis are those having extremely low or high GC content (less than 25% and greater than 75%), homopolymeric runs of 10 or more As and Ts or 6 or more Gs and Cs. Other structural motifs such as repeats or hairpins may also influence our ability to construct gBlocks Gene Fragments. The decision to accept or reject an order for a particular gBlocks fragment sequence is complex and multifactorial. All of our gBlocks fragments are reviewed by our Gene Specialists prior to manufacturing to ensure the sequences can be made successfully. Questions about gBlocks Gene Fragments? Contact our Gene Specialists directly at genes@idtdna.com.

  • What is the maximum size construct that can be assembled with gBlocks® Gene Fragments using Gibson Assembly®?

    The original paper describing the Gibson Assembly® Method [Gibson et al., 2009] suggests the method can be used to assemble constructs in the size range of small bacterial genomes, and megabase assemblies have been demonstrated using this protocol. However, at IDT we have limited our gBlocks fragment assemblies to 2–2.3 kb gene constructions using 4–5 gBlocks Gene Fragments using the Gibson Assembly Master Mix, available from New England BioLabs. For larger assemblies, we recommend a sub-assembly strategy with sequence-verification of the component parts.

    Reference: Gibson DG, Young L, et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6 (5):343–345. doi:10.1038/nmeth.1318. PMID 19363495.

  • Can I order duplexed Ultramer® Oligonucleotides?

    We unfortunately do not offer duplexed Ultramer Oligonucleotides. However, we do provide gBlocks Gene Fragments, which are sequence-verified, dsDNA fragments up to 2000 bp. Alternatively, you can order single-stranded Ultramer Oligonucleotides and anneal them using the following Annealing Protocol: Dissolve the oligos at high concentration—as high as 500 µM if possible, although as low as 100 µM will work;  i.e., 1-10 OD260 units / 100 µL—in STE Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA) or Nuclease-free Duplex Buffer (30 mM Hepes pH 7.5, 100 mM KAc) (available from IDT). The presence of some salt is necessary for the oligos to hybridize. Then mix the two oligos in equimolar concentrations, heat to 94° C, and allow the solution to cool slowly to room temperature. For sequences with significant hairpin potential, a more gradual cooling step is beneficial; this is easily done by placing the oligos in a water bath or heating block and unplugging the machine. The resulting product will be a stable, double-stranded construct.