Assembly Method

Select the assembly method you want to use

Description of Cloning Method

BioBricks is a digestion-ligation based binary assembly method. The version of the method applied here (BBF RFC 10) requires that all parts and vectors are preceded by EcoRI and XbaI (EX) sites, and followed by SpeI and PstI (SP) sites. Digesting these restriction sites produces common sticky ends, which allow 2 BioBricks parts and their cloning vector to be assembled.

ASSUMPTIONS: It is assumed that cloning vector pSK1A2 is used for all steps and stages and that inserts are digested and gel extracted for each cloning step.

Assembly Efficiency
Parts per ReactionEfficiency
2
Description of Cloning Method

CPEC (Circular Polymerase Extension Cloning) is a scarless multi-way assembly method that does not require digestion, ligation or homologous recombination. Multiple double stranded DNA fragments with overlapping flanking regions are denatured and amplified using DNA polymerase. The version of the method applied here adds NotI sites at the end of PCR fragments if intermediate clones are required. These fragments can then be extracted and used in the next cloning stage. The version of the method applied here, if there are solutions that require more than one stage, intermediate vectors are designed with homology to regions flanking these intermediates for the next step. In between that homology region, there is an inserted NotI site so that these intermediates may be cleaved and used in the next stage. These intermediate fragments are then extracted and used in the next cloning stage.

ASSUMPTIONS: It is assumed that cloning vector pSK1A2 is used for all steps and stages and that inserts for intermediate steps are digested with NotI and gel extracted for the subsequent cloning stage.

Assembly Efficiency
Parts per ReactionEfficiency
2
3
4
5
6
efficiency value efficiency value
Description of Cloning Method

Gibson is an isothermal, scarless multi-way assembly method that leverages homologous recombination. Gibson assembly relies on the combined activities of a T5 exonuclease, DNA polymerase, and a DNA ligase to assemble DNA molecules with overlapping flanking regions. In the first part of the reaction, the 5' ends of each DNA fragment is chewed-back by the exonuclease, allowing homologous single-stranded regions to anneal. The gaps on each DNA strand is then repaired with DNA polymerase and DNA ligase to leave a final clone. The version of the method applied here, if there are solutions that require more than one stage, intermediate vectors are designed with homology to regions flanking these intermediates for the next step. In between that homology region, there is an inserted NotI site so that these intermediates may be cleaved and used in the next stage. These intermediate fragments are then extracted and used in the next cloning stage.

ASSUMPTIONS: It is assumed that cloning vector pSK1A2 is used for all steps and stages and that inserts for intermediate steps are digested with NotI and gel extracted for the subsequent cloning stage.

Assembly Efficiency
Parts per ReactionEfficiency
2
3
4
5
6
efficiency value efficiency value
Description of Cloning Method

Gateway-Gibson is a hybrid method for building transcriptional units from promoter and gene libraries with Gateway assembly and assemble them hierarchically with Gibson assembly by cleaving intermediates with I-SceI and using them in Gibson reactions.

ASSUMPTIONS: It is assumed that coded destination vector backbone pSK1A2 is is used for all Gibson steps and stages and that the coded destination vector backbone pSB1K3 is used for all Gateway steps. It is also assumed that for both types of destination vectors that the appropriate L/R Gateway sites are cloned into the backbones independently with flanking BsaI sites compatible insert sequences. Raven algorithms will force the construction of transcriptional units with Gateway for this method. These are identified with the "promoter" and "gene" keywords. All pieces in between a promoter and a gene will become an additional piece for the Gateway reaction. Parts that fall outside of transcriptional units will be made into independent Gibson clones in the completed construct.

*** WARNING *** This assembly method is still undergoing testing! Do not use this method in practice at this time. The final version will be available shortly. Please contact the developers with questions.

Assembly Efficiency
Parts per ReactionEfficiency
2
3
4
5
6
7
8
efficiency value efficiency value
Description of Cloning Method

Golden Gate is a scarless multi-way assembly method that relies on Type IIs restriction enzymes. The version of the method applied here uses a tiered cloning system, which alternates the use of restriction enzymes BbsI and BsaI in every other stage to cleave inserts from donor plasmids. This version also adds SpeI sites at the end of LacZ PCR fragments used to make destination vectors. Fusions sites are selected as a 4-bp combination of adjacent parts in a cloning reaction. This method assumes the acquisition of the LacZ fragment necessary for blue-white screening and the vector pSK1A2.

ASSUMPTIONS: It is assumed that cloning vectors pSB1A2 and pSB1K3 are used for alternating cloning stages and that the LacZ fragment necessary for blue-white screening is acquired and used to make destination vectors.

Assembly Efficiency
Parts per ReactionEfficiency
2
3
4
5
6
efficiency value efficiency value
Description of Cloning Method

MoClo is a multi-way assembly method that uses common overhang junctions generated by type IIs restriction enzymes. The version of the method applied here (BBF RFC 94) uses a tiered cloning system, which alternates the use of restriction enzymes BbsI and BsaI in every other stage to cleave inserts from donor plasmids. This version also adds SpeI sites at the end of LacZ PCR fragments used to make destination vectors. Although overhang junctions add a 4-bp assembly scar between parts, the use of common junctions can be used to maximize part reuse. This method assumes the acquisition of the LacZ fragment necessary for blue-white screening and the vector and pSB1K3.

ASSUMPTIONS: It is assumed that cloning vectors pSB1A2 and pSB1K3 are used for alternating cloning stages and that the LacZ fragment necessary for blue-white screening is acquired and used to make destination vectors.

Assembly Efficiency
Parts per ReactionEfficiency
2
3
4
5
6
efficiency value efficiency value
Description of Cloning Method

SLIC (Sequence and Ligation Independent Cloning) is a scarless multi-way assembly methods that leverages in vitro homologous recombination and single-strand annealing. SLIC uses exonuclease to generate single stranded DNA overhangs in inserts and cloning vectors. SLIC effectively mimics in vivo homologous recombination. The version of the method applied here, if there are solutions that require more than one stage, intermediate vectors are designed with homology to regions flanking these intermediates for the next step. In between that homology region, there is an inserted NotI site so that these intermediates may be cleaved and used in the next stage. These intermediate fragments are then extracted and used in the next cloning stage.

ASSUMPTIONS: It is assumed that cloning vector pSK1A2 is used for all steps and stages and that inserts for intermediate steps are digested with NotI and gel extracted for the subsequent cloning stage.

Assembly Efficiency
Parts per ReactionEfficiency
2
3
4
5
6
efficiency value efficiency value

Constructs

Select the plasmids you want to construct

Available Plasmids to Construct
Selected Plasmids


Primers

Specify your requirements for primers that will be designed for your assembly

Oligo Name Prefix
Target Homology Tm
Target Homology Length
Minimum PCR Length
Minimum Cloning Length
Maximum Primer Length

Run

Summary of your design parameters

A summary of your assembly plan will appear here