repliQa HiFi Assembly Mix
Features & Benefits
- Formulated to increase number of transformants
- Assemble up to 6 fragment inserts without the need for restriction enzymes
- Flexibility in design with 10x MM enabling assembly of low concentration DNA samples
- Eliminates dilution step resulting in easier workflow and 1 hr assembly
- Includes DpnI to reduce background when using plasmid templates for PCR
repliQa HiFi Assembly Mix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
The repliQa™ HiFi Assembly Mix simplifies the construction of recombinant DNA through seamless assembly of multiple DNA fragments in a single, isothermal reaction. Similar in principle to the Gibson Assembly® Method, the high efficiency repliQa HiFi Assembly Mix is ideal for a range of genetic engineering applications including:
- Routine molecular cloning
- Site-directed mutagenesis
- Synthetic biology
- Construction of libraries for directed evolution studies
The concentrated (10x), two component format allows flexibility in design of assembly reactions and compatibility with less concentrated DNA samples. The repliQa Mix has been optimized for use with a total input quantity of DNA fragments in the range of 0.03 to 0.5 pmols. Assembly of up to five DNA fragments is recommended, though the repliQa Mix has been used successfully for more complex assemblies. The mix supports assembly of multiple DNA fragments in a single 1-hr reaction.
High efficiency cloning from low DNA inputs
repliQa HiFi Assembly Mix increases transformation efficiency without the need for diluting or purifying the assembly reaction prior to transformation of competent cells, resulting in less hands-on- time and faster workflows.
Assemble larger number of fragments
repliQa HiFi Assembly Mix allows for large inserts to be cloned at a very high efficiency resulting in significantly more positive clones eliminating the need to repeat experiments due to erroneous or insufficient clones.
The repliQa™ HiFi Assembly Mix simplifies the construction of recombinant DNA through scarless assembly of multiple DNA fragments in a single, isothermal reaction.
Dilution of the assembly reaction not required – high transformation efficiency, less hands-on time
Three DNA fragments containing 23 bp overlaps were generated by PCR, DpnI treated umn purified.
The three fragments, 4.2 kb, 3.1 kb, and 400 bp in size, were combined in a 1:1.4:5 molar ratio. Total DNA quantities used are indicated (x-axis) and reacted at 50 °C for 60 minutes according to the protocol. One microliter of the undiluted assembled products or one microliter of a 4-fold dilution of the assembled products was used to transform 30 μl of chemically competent cells.
High transformation efficiency greater number of fragments
PCR fragments containing 30 bp overlaps were DpnI treated, purified, and assembled according to the protocol.
PCR fragments containing 30 bp overlaps were DpnI treated, purified, and assembled according to the protocol. Reactions contained the indicated number of DNA fragments (0.1 pmol each) and were incubated at 50 °C for 60 minutes. 1 ul of the reactions were used to transform 30 µl of chemically competent cells.
repliQa HiFi Assembly Enzyme Mix Optimized formulation of enzymes for 5’-end resection, high fidelity 3’- end extension, and nick sealing.
repliQa 10X Assembly Reaction Buffer 10X reaction buffer containing dNTPs, magnesium, and cofactors
DpnI (20 U/μl) Restriction endonuclease for the (optional) post- PCR digestion of residual unamplified plasmid template.
Storage & Handling
Store kit components in a constant temperature freezer at -25°C to -15°C upon receipt. For long term buffer storage (> 30 days) store buffer at -70°C.
Refer to the product label or lot-specific Product Specification Sheet (PSF) for applicable expiration date.
Related ResourcesProduct FlyersProduct ManualsTechnical NotesCofA (PSF)PSF-95190-010-Lot#021291PSF-95190-010-Lot#023986PSF-95190-050-Lot#023987PSF-95190-010-Lot#024172PSF-95190-050-Lot#024173PSF-95190-010-Lot#024898PSF-95190-050-Lot#024902PSF-95190-050-Lot#025616PSF-95190-010-Lot#025922PSF-95190-050-Lot#025928PSF-95190-010-Lot#026937PSF-95190-050-Lot#027074PSF-95190-050-Lot#027848PSF-95190-010-Lot#028645PSF-95190-010-Lot#029212PSF-95190-050-Lot#030311PSF-95190-010-Lot#030391PSF-95190-010-LOT#66141034PublicationsEngineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCRClick here to see all PublicationsRyan C. Heller, Nucleic Acids Research - 2019AbstractReverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5′→3′ nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.FAQsWhat is the recommended amount of sequence overlap?The repliQa HiFi Assembly Mix is optimized for assembly of fragments with overlap regions between 15 – 60 bp.Is it necessary to purify the PCR fragments that will be used in the assembly reaction?Purification of PCR product using a spin column-based cleanup or other method is not required but is highly recommended to achieve highest efficiency fragment assembly.What are the advantages of the repliQa HiFi Assembly Mix for cloning?The repliQa HiFi Assembly Mix allows simultaneous cloning of multiple insert fragments into vectors without creating scars. In contrast to traditional cloning, there is no requirement for restriction endonucleases and unique restriction sites.Is there a preferred method for transforming E.coli with the assembled DNA?The products of the repliQa HiFi Assembly reaction can be introduced into E.coli either by chemical transformation or electroporation. If electroporation is to be used for transforming cells, we recommend first diluting the assembly reaction 1:5 in high purity water.Click here to see all FAQsWhen should the DpnI be used?If plasmid DNA is used as PCR template for generation of fragments for assembly, it is recommended to treat the PCR reactions with DpnI prior to the assembly reaction to eliminate residual methylated plasmid that could give rise to colonies lacking the assembled product of interest.