gBlocks^® Gene Fragments

gBlocks Gene Fragments in plates

For projects that require large quantities of gBlocks Gene Fragments (≥48 fragments at a time), delivery in plates may be a more cost-effective option.

gBlocks Gene Fragment Libraries

gBlocks Gene Fragment Libraries are pools gBlocks fragments that are ideal for demanding applications such as generating recombinant antibodies or protein engineering. While they can be used directly in applications such as In vitro transcription, or as standard or internal controls for qPCR or NGS experiments, they are most often assembled into functional genes or longer constructs by seamless assembly techniques or traditional restriction cloning. With gBlocks Gene Fragments Libraries, you can generate hundreds of thousands of constructs at a fraction of the cost that creating multiple variant libraries would require.

Ordering gBlocks libraries is just as easy as ordering standard gBlocks Gene Fragments. The tool’s interface allows N or K (G or T) mixed bases; however, they must be entered in the appropriate format. gBlocks Gene Fragments in the libraries can be 251 to 500 bp in total length, including the variable region. The variable regions can be up to 18 consecutive N or K bases long, and must be at least 125 bp from either end of the gene fragment (Figure 1).

Need something else? Tell us about it! We realize that we have a limited offering for gBlocks Gene Fragments Libraries. We will be offering more complex libraries in the future. Help us prioritize our product development by sharing what you need from us with as much detail as possible (i.e., show us sequences, including features such as mixed bases, annotations, and drawings) and send to libraries@idtdna.com.

Why do we limit the variable regions to 18 mixed bases?

When you order a gene fragment library that contains 18 "N" mixed bases, you order a pool of 418 gene fragments—about 68.7 billion different gBlocks Gene Fragments. Increasing the number of variable bases beyond 18 would result in more sequence combinations than can be accommodated in the 200 ng of product provided, which would compromise the overall representation of sequences within the pool.

Quality assurance

Every gBlocks Gene Fragment you receive goes through the following verification process:

1. Sequences (125–3000 base pairs) are entered on the gBlocks Gene Fragments order entry page. A, C, G, T bases, as well as up to 18 consecutive N or K variable bases can be entered. Other mixed degenerate bases are currently not available, and neither are “modified" bases (inosine, uridine).
2. Upon order entry, the entered sequences are reviewed by both our automated screening tools and our scientists to identify characteristics that may interfere with synthesis.
3. Biosafety and regulatory conformance check:
   • A biohazard disclosure statement is required for each gene order.
   • IDT reserves the right to refuse any order that does not pass this analysis. If one of your sequences does not pass the screen criteria, you will be contacted by a gene services specialist to determine the best way to proceed.

Quality control and sequence verification

Each gBlocks Gene Fragment undergoes the following quality control procedures:

1. Size verification by capillary electrophoresis
2. Sequence identification by mass spectrometry

This rigorous testing ensures that, in the majority of cases, >80% of recombinant colonies obtained from cloning each gBlocks Gene Fragment will contain the desired insert. More complex sequences may suffer from reduced fidelity, which can be addressed by the end user sequencing additional clones.

For gBlocks Gene Fragment Libraries, each constant region is verified as above. The final library product is size verified by capillary electrophoresis.

High fidelity gene fragments simplify the cloning process

gBlocks Gene Fragments possess the high sequence fidelity and DNA purity (Figure 1) necessary for synthetic biology and gene assembly methods.

Easy gene assembly using gBlocks Gene Fragments

One or more gBlocks Gene Fragments can be easily assembled using the Gibson Isothermal^® Assembly to generate larger DNA sequences [1]. The fidelity of gBlocks fragments substantially reduces the number of clones that need to be sequenced to identify the intended construct when compared to other methods of gene assembly using DNA oligonucleotides. Table 1 shows the average frequency of 100% correct sequences observed after cloning 1 or more fragments using a method similar to the Gibson Assembly method. Note that assembly methods such as blunt cloning and restriction cloning consistently demonstrate lower fidelity.

Table 1. Proportion of clones with 100% correct sequences observed from cloning 1–4 gBlocks Gene Fragments.

Compatibility with traditional cloning methods and vectors

gBlocks Gene Fragments are compatible with all cloning methods that require double-stranded DNA as a starting material, allowing easy assembly of your desired construct sequence into your favorite cloning system.

Table 2. gBlocks Gene Fragments are compatible for cloning in a variety of standard vectors, using common cloning methods.*

* Single gBlocks Gene Fragments ranging between 223 and 296 bp were cloned into pUC57, pBluescript II, pET27, psiCHECK-2, Zero Blunt TOPO, pIDTSMART, and pGEM T Easy plasmids. Resulting clones were sequence verified by double-stranded sequencing.

References

1. Gibson D, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.

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Codon Optimization Tool

Optimizes a protein coding sequence that is derived from one species for expression in another.

Genes & gene fragments order status

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gBlocks Gene Fragments Entry Tool