Megamer® Single-Stranded DNA Fragments 

Megamer Single-Stranded DNA Fragments are sequence-verified, single-stranded DNA that range in length from 201–2000 bases. They can be used in applications such as homology-directed repair of CRISPR-mediated genome editingin vitro transcription, and more.

  • Synthesized with clonally purified DNA, which offers the greatest purity available
  • Sequence-verified via next generation sequencing
  • Typically shipped within 20 business days

Megamer Single-Stranded DNA Fragments (paired) 

Includes your ssDNA fragment of interest and the complementary strand, delivered dry in separate tubes. Fragments are normalized to 3 µg final yield. Both strands will be delivered for each sequence entry. 
ProductPrice
Megamer™ ssDNA Fragments—Sense + Antisense, 201–500 bases$860.00 SGD
Megamer™ ssDNA Fragments—Sense + Antisense, >500 bases$1.70 SGD / base

Megamer Single-Stranded DNA Fragments (individual)

Includes your ssDNA fragment of interest, without the complementary strand, delivered dry. Fragments are normalized to 3 µg final yield. 
ProductPrice
Megamer™ ssDNA Fragment, 201–500 bases$645.00 SGD
Megamer™ ssDNA Fragment, >500 bases$1.30 SGD / base

Megamer ssDNA Fragments are single-stranded genomic blocks for applications such as homology-directed repair of CRISPR-mediated genome editingin vitro transcription, and more. They range in length from 201–2000 bases and are synthesized with clonally purified DNA, which offers the greatest purity available. Megamer ssDNA Fragments are sequence-verified via next generation sequencing and are typically shipped within 20 business days*. They are composed of A, T, G, and C nucleotides only. 

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


* The time it takes to manufacture a Megamer ssDNA Fragment is dependent on many factors and, in a few cases, may exceed the estimated delivery time.
 

1. 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 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

Integrated DNA Technologies screens every Megamer ssDNA Fragment for potentially dangerous pathogen sequences, and to verify that IDT’s 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.