Synthetic Biology

NEW! Introducing gBlocks® Gene Fragments Libraries

Using gBlocks® Gene Fragments as Synthetic Templates for qPCR

gBlocks® Gene Fragments 

Cheaper, faster, better synthetic biology

gBlocks® Gene Fragments are double-stranded, sequence-verified genomic blocks that ship in only a few working days for affordable and easy gene construction or modification, applications such as antibody research and CRISPR-mediated genome editing, use as qPCR standards, and more.

gBlocks® Gene Fragments Libraries are pools of gBlocks Gene Fragments that contain up to 18 consecutive variable bases (N or K) for recombinant antibody generation or protein engineering.

Sequence information is always secure and confidential at IDT. Non-disclosure agreements are available through IDT legal services upon request.



New! N and K mixed bases are now accepted by the ordering tool to form gBlocks® Gene Fragments Libraries.

DescriptionPricing
gBlocks® Gene Fragments 125-500 bp$98.11 USD
gBlocks® Gene Fragments 501-750 bp$135.37 USD
gBlocks® Gene Fragments 751-1000 bp$160.21 USD
gBlocks® Gene Fragments 1001-1250 bp$222.30 USD
gBlocks® Gene Fragments 1251-1500 bp$267.01 USD
gBlocks® Gene Fragments 1501-1750 bp$309.23 USD
gBlocks® Gene Fragments 1751-2000 bp$353.94 USD
Product
Shipped (BD)1
gBlocks® Gene Fragments 125-500 bp 2–4
gBlocks® Gene Fragments 501-750 bp
2–4
gBlocks® Gene Fragments 750-1000 bp 3–5
gBlocks® Gene Fragments 1001-1250 bp
5–8
gBlocks® Gene Fragments 1251-1500 bp
5–8
gBlocks® Gene Fragments 1501-1750 bp
5–8
gBlocks® Gene Fragments 1751-2000 bp
5–8
Single Mixed Base
10–15
>1 Mixed Bases (each)
10–15

1Business Days: shipping time for gBlocks Gene Fragments is dependent on length and complexity, and in a few cases may exceed these estimated times.

Single Mixed Base
> 1 Mixed Bases (each)

$173.87 USD
$86.93 USD / Base

Product Specifications

gBlocks Gene Fragments

  • Available in lengths of 125–2000 bp
  • A, T, C or G residues only 
  • Delivered dry, normalized to 200 ng

gBlocks Gene Fragments Libraries

  • Available in lengths of 251–500 bp 
  • Up to 18 consecutive N or K residues 
  • Delivered dry, normalized to 200 ng

gBlocks® Gene Fragments

gBlocks Gene Fragments are double-stranded DNA molecules of 125–2000 bp in length. gBlocks Gene Fragments are synthesized using the same industry-leading, high-fidelity synthesis chemistry developed by IDT for our Ultramer® oligonucleotides, and are sequence verified prior to shipping. The high sequence fidelity and rapid delivery time make gBlocks Gene Fragments ideal for a range of synthetic biology applications, including the ability to easily assemble multiple gene fragments to reliably generate larger gene constructs.

  • Highly versatile—gBlocks Gene Fragments can be used to easily and reliably assemble almost any sequence, and are compatible with most published cloning methods, including the Gibson Assembly® Method, and blunt-end or cohesive-end cloning protocols.
  • Easy isothermal gene assembly—Using the Gibson Assembly Method, multiple gBlocks Gene Fragments can be assembled into a larger gene construct, in a single reaction that takes about 1 hour. A simple, 20–80 nt sequence overlap is required when designing the gene fragments for assembly.
  • Affordable—gBlocks Gene Fragments are up to half the price of other synthetic gene constructs, making synthetic biology accessible to any lab.
  • Short delivery time—gBlocks Gene Fragments are typically shipped within 5 business days (up to 1kb) or 8 business days (up to 2kb).
Synthetic Biology Partners

Assembly and cloning of multiple gBlocks Gene Fragments is quick and easy with New England Biolab’s Gibson Assembly™ Master Mix

NEB Gibson Assembly Master Mix

Synthetic Genomics logo

IDT and SGI are working together to construct custom, synthetic, double-stranded nucleic acids up to 5 kb. IDT uses its custom oligonucleotide manufacturing expertise, including its recently launched gBlocks Gene Fragments product line, in combination with SGI’s proprietary technologies to efficiently assemble small gene constructs.

SGI Gibson Assembly

gBlocks® Gene Fragments Libraries

gBlocks Gene Fragments Libraries are pools of gBlocks Gene Fragments up to 500 bp in length that contain up to 18 consecutive variable bases (N or K). They are ideal tools for demanding applications such as generating recombinant antibodies or protein engineering. 

Below is a representation of the general ordering format for gBlocks Gene Fragments Libraries

Need something else? Tell us about it! 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.

Ordering format for gBlocks Gene Fragments Libraries

gBlocks Gene Fragments Libraries are entered the same way as gBlocks Gene Fragments. The ordering interface now accepts N or K (G or T) mixed bases when present in an acceptable format (K mixed bases placed in the third position of codons eliminate two of the three stop codons from being included in the gene fragments libraries). The gBlocks Gene Fragments 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 needs to be at least 125 bp from either end of the gene fragment.

Why did IDT 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.

How to use gBlocks Gene Fragments Libraries

gBlocks Gene Fragments Libraries can be used directly in some suitable applications such as In vitro transcription or when used as standard or internal controls for qPCR or NGS experiments. More often, they are assembled into functional genes or longer constructs by seamless assembly techniques or traditional restriction cloning.

Adding diversity at reasonable cost

For screening purposes, gBlocks Gene Fragments Libraries allow for the generation of hundreds of thousands of constructs at a fraction of the cost of generating variant libraries.

Quality Assurance

Each gBlocks Gene Fragment ordered from IDT will go through the following verification process:

  1. Researchers enter the gene fragments sequences (125–2000 base pairs) 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 not currently available; no “modified" bases (Inosine, Uridine).
  2. Automated review by screening tools at the time of order entry and, and expert review by IDT scientists, of entered sequences for 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.

QC procedure 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
  3. Final consensus sequence verification

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 have reduced fidelity, which can be addressed by the end user sequencing additional clones.

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

Guaranteed yield

  • gBlock Gene Fragments are delivered dry, normalized to 200 ng.

Confidentiality

  • All sequence information is kept confidential at IDT.
  • All online ordering steps, including sequence entry and choice of parameters, are also secure and protected.

Possible applications of gBlocks Gene Fragments include, but are not limited to:

  • Protein Expression
    • Recombinant antibodies
    • Novel fusion proteins
    • Codon optimized short proteins
    • Functional peptides: catalytic, regulatory, binding domains
  • microRNA genes
  • Template for in vitro transcription (IVT)
  • Regulatory sequence cassettes
  • Microarray-ready cDNA
  • Gene variants and SNPs
  • DNA vaccines
  • Standards for quantitative PCR and other assays
  • Functional Genomics
    • Limitless flexibility for protein mutagenesis
      • Mutant libraries
      • Deletion mutants

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.

High fidelity gene fragments simplify the cloning process

gBlocks® Gene Fragments possess the high sequence fidelity and DNA purity necessary for synthetic biology and gene assembly methods.

To demonstrate the fidelity, 43 different gBlocks Gene Fragments, ranging in length from 126–459 bp with CG ratios between 40 and 70%, were synthesized and blunt cloned into pIDTSMART-Amp. For each gBlocks Gene Fragment, fidelity was determined by Sanger sequencing of multiple cloned inserts.

The results demonstrate that on average, 90% of gBlocks Gene Fragments tested, when directly cloned, were the correct sequence. Of the 43 gBlocks Gene Fragments sequences tested, 22 produced inserts that were correct in >90% of the selected clones, and 40 gBlocks Gene Fragments produced inserts that were correct in >80% of the selected clones (Figure 1). In the remaining 3 fragments, more than half of the inserts had the correct sequence. This means that, with gBlocks Gene Fragments, you will be able to quickly generate, and identify your desired DNA constructs, while saving time and money. 

Figure 1. gBlocks® Gene Fragments make it easy to produce the correct synthetic genes. Forty-three gBlocks Gene Fragments ranging from 126–459 bp and with CG ratios between 40 and 70% were synthesized and cloned into pIDTSMART-Amp by blunt-end cloning. Ligated plasmids were transformed into XL1Blue cells, and between 3 and 25 clones for each gBlocks Gene Fragment were selected and sequenced, using traditional Sanger sequencing. The average sequence fidelity for all sequences was 90%, and 40 out 43 gBlocks Gene Fragments produced >80% of inserts with the correct sequence.

2. Easy gene assembly using multiple gBlocks Gene Fragments

Two or more gBlocks Gene Fragments can be easily assembled using the Gibson Isothermal® Assembly to generate larger DNA sequences [1]. The fidelity of gBlocks Gene 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.

The EGFP and kanamycin genes were assembled from either 2 or 3 gBlocks Gene Fragments, respectively. Each gene was divided into approximately equal size gBlocks Gene Fragments with 30 bp sequence overlaps at the ends of adjacent fragments as is required for the Gibson Assembly method [1]. The fragments were then assembled into a pUC57 vector. The results show that in most cases, when assembling 2–3 gBlocks Gene Fragments, sequencing as few as eight clones is sufficient to find a wild-type sequence; more challenging sequences and complex designs may require sequencing of additional clones. For more information selecting clones, see Tips from the Bench below.

Table 1. Multiple gBlocks Gene Fragments® can be assembled using the Gibson Assembly® method to easily generate larger, high-fidelity DNA sequences.

Wild-type Clones  Gene length (nt)  # gBlocks Gene Fragments   Correct clones*
EGFP  720   2  3/8
Kanamycin 816 3 2/8

* Verified by double-stranded DNA sequencing

Experimental Details: Kanamycin and EGFP genes were assembled from 2 or 3 gBlocks Gene Fragments, respectively, and cloned into a pUC57 vector, linearized with EcoRV, using the Gibson Assembly™ method [1]. Isothermal assembled plasmids were transformed into XL1Blue cells and resulting clones were verified for the assembled genes, using traditional Sanger sequencing.

Tips from the Bench

gBlocks Gene Fragments are produced using our highest fidelity synthesis methods. However, the chance of a single error affecting a final assembled molecule increases with the number of fragments assembled. We recommend sequencing at least 2 x (number gBlocks fragments assembled) clones to give you the highest probability of successfully identifying your desired target. For example, if you assemble 4 gBlocks Gene Fragments we recommend sequencing 8 clones to have the best chance (95%) of obtaining your desired construct.


3. gBlocks Gene Fragments are compatible with traditional cloning methods and vectors

Because gBlocks Gene Fragments are compatible with all cloning methods requiring double-stranded DNA as a starting material, they have exceptional potential for designing and assembling your desired construct sequence into any favorite cloning system.

Table 2. gBlocks Gene Fragments are compatible for cloning in a variety of standard vectors, using common cloning methods (traditional blunt-end cloning and Gibson Assembly® methods are shown in Sections 1 and 2 above, respectively).

  Correct clones   Clones sequenced % wild-type clones 
Restriction cloning   27 32   84% 
TOPO cloning   3 4 75%

Experimental Details: Single gBlocks Gene Fragments ranging between 223 and 296 bp were cloned into selected plasmids, including: pUC57, pBluescriptII, pET27, psiCHECK-2, Zero Blunt TOPO, pIDTSMART, pGEM T Easy. 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.

gBlocks User Guide

The guide contains protocols for cloning of gene fragments into plasmids for functional use, and troubleshooting techniques.

Download the gBlocks User Guide

gBlocks Gene Fragments Protocols

The following protocols are recommended methods for assembling and amplifying gBlocks Gene Fragments. Handling and storage instructions are also included.

Gene Synthesis Design Considerations

Highly versatile gBlocks Gene Fragments can be easily assembled and cloned into the vector of your choice using a wide variety of cloning methods, including the Gibson Assembly® method, blunt-end and cohesive-end cloning protocols. For added flexibility, gBlocks Gene Fragments can be ordered with or without a 5’-phosphate group; select the correct phosphorylation option based on cloning method.

Assembly Method gBlock Gene Fragment Phosphorylation Note
 Isothermal Assembly  Un-Phosphorylated Requires a simple 20–80 bp overlap in DNA elements being joined. Can be used to join multiple gBlocks Gene Fragments in a single reaction.
 Restriction Cloning  Un-Phosphorylated Consider adding 6–8 nucleotides on each end of your gBlocks Gene Fragment to ensure efficient restriction digestion
 TOPO Cloning  Un-Phosphorylated For T/A cloning, gBlocks Gene Fragment needs to be adenylated using a Taq polymerase in presence of dATP
 Blunt-end Cloning  5’-Phosphorylated Linearized vector must be 5’-dephosphorylated

Assemble gBlock Gene Fragments using Isothermal Assembly

Isothermal Assembly: Quick, Easy Gene Construction — DECODED Newsletter

The isothermal assembly method, recently developed by Gibson, greatly simplifies the process for molecular cloning of synthesized DNA molecules. Isothermal assembly also makes it possible to include larger, more complex assemblies than traditional cloning methods.

View Article

Quick Protocols

These Quick Protocols are provided as a reference for users experienced with molecular biology techniques and cloning. Expanded instructions are included in the gBlocks User Guide.

Cohesive-End Cloning

Cohesive-end cloning is highly efficient due to the hydrogen bond stabilization of the complementary DNA overhangs that are created by many restriction digests.

Heading

Required

  • gBlocks Gene Fragments with non-phosphorylated ends and include the restriction sites at least 5 bases internal from the ends
  • Vector containing the appropriate restriction site(s)
  • Desired restriction endonuclease(s)
  • Alkaline phosphatase
  • T4 DNA Ligase
  • Competent cells

Procedure

  1. Linearize 1 μg of vector by restriction digest.
  2. Remove the 5’ phosphates from the vector with an alkaline phosphatase.
  3. Gel purify the linearized vector.
  4. Resuspend the gBlocks Gene Fragments in 20 μL of TE (10 mM Tris, 1 mM EDTA, pH 7.5).
  5. Prepare ends of gBlocks Gene Fragments by restriction digest of 10μL.
    Note: If chosen restriction enzyme cannot be heat inactivated, column purify and elute in 10–20 μL following manufacturer’s instructions
  6. Place the following in the ligation reaction:
    1. 20ng gBlocks Gene Fragments
    2. 50 ng of vector from step 3
    3. 400 cohesive end units of T4 DNA ligase
    4. Fresh T4 DNA ligase buffer diluted to 1X
    5. Add H2O to final volume 20 μL 
  7. Incubate for 2 hours at 16°C.
  8. Transform 2 μL onto competent cells following the manufacturer’s instructions.
 View Cohesive-End Cloning Protocol

Blunt-End Cloning

Blunt-end cloning is also a popular cloning method. However, its efficiency is typically 50- to 100-fold lower than cohesive end cloning, and it is non-directional [1]. Due to the poor efficiency and limited starting material, we do not generally recommend blunt-end cloning of gBlocks Gene Fragments by non-experienced users.

Heading

View Blunt-End Cloning Protocol

Required:

  • gBlocks Gene Fragments with phosphorylated ends
  • Vector containing the appropriate restriction site
  • Desired restriction endonuclease (RE)
  • Alkaline phosphatase
  • T4 DNA Ligase
  • Competent cells

Procedure:

  1. Linearize 1 μg of vector using a blunt-cutting RE such as EcoRV or MscI.
  2. Remove the 5’ phosphates from the vector with an alkaline phosphatase.
  3. Gel purify the linear fragment of the vector digest.
  4. Resuspend the gBlocks Gene Fragments in 20 μL of TE (10 mM Tris, 1 mM EDTA, pH 7.5).
  5. Place the following in the ligation reaction:
    1. 4 μL gBlocks Gene Fragments (40 ng)
    2. 50 ng of vector from step 3
    3. 400 cohesive end units of T4 ligase
    4. Fresh T4 DNA ligase buffer diluted to 1X
    5. Add H2O to final volume 20 μL
  6. Incubate for 2 hours at 16°C.
Transform 2 μL into competent cells following the manufacturer’s instructions.

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.

gBlocks Gene Fragments Biosecurity

Integrated DNA Technologies screens the sequence of every gBlocks Gene Fragment order that is received to identify regulated and other potentially dangerous pathogen sequences, and to verify that IDT’s gene customers are legitimate scientists engaged in beneficial research.

IDT is among the five founding members of the International Gene Synthesis Consortium (IGSC), and helped to create the IGSC’s Harmonized Screening Protocol. The Harmonized Screening Protocol describes the gene sequence and customer screening practices that IGSC member companies employ to prevent the misuse of synthetic genes. IDT takes the steps set out in the Harmonized Screening Protocol to screen the sequences of ordered genes and the prospective customers who submit those orders.

For more information about the IGSC and the Harmonized Screening Protocol, please visit the website at http://www.genesynthesisconsortium.org/.

In October of 2010, the United States government issued final Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA, describing how commercial providers of synthetic genes should perform gene sequence and customer screening. IDT and the other IGSC member companies supported the adoption of the Screening Framework Guidance, and IDT follows that Guidance in its application of the Harmonized Screening Protocol. For more information, please see 75 FR 62820 (Oct. 13, 2010), or http://federalregister.gov/a/2010-25728.