Updated to allow for in vitro transcription with both SP6 and T7 promoters
BsaI-site removed from ampicillin-resistant gene (allows for cloning of Golden Gate Assembly modules)
More restriction sites added, including four 8-base cutters
This PCR Cloning Kit contains an optimized Cloning Mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low background. It allows simple and quick cloning of any PCR amplicon, whether the amplification reactions are performed with proofreading DNA polymerases, such as Q5® which produce blunt ends; or nonproofreading DNA polymerases, such as Taq or Taq mixes (OneTaq®, LongAmp®Taq) which produce single base overhangs. This is possible due to “invisible” end polishing components in the master mix that are active during the ligation step only if needed. The kit also allows direct cloning from amplification reactions without purification, and works well whether or not the primers used in the PCR possess 5´-phosphate groups.
The ultimate in flexibility: clone with any amplicon made with any DNA polymerase, with or without 5´ phosphates, purified or not!
PCR cloning with low/no backgroundA 500 bp PCR product incubated with the linearized vector in a 3:1 ratio according to recommended protocol. 2 μl of reaction was transformed into provided NEB 10-beta Competent E. coli and 1/20th of the outgrowth was plated. Left plate serves as the control, with vector backbone only, right plate contains PCR insert.Cloning Kit Protocol Overview pMiniT 2.0 Vector Map
Map shown above displays the construct formed if no insert is present. Unique restriction sites are shown
in black. Additional restriction sites that can be used for subcloning are shown in red. Expanded box below
shows location of sequencing primers, restriction sites for subcloning, and placement of insertion site
within the toxic minigene.
This product is related to the following categories:
The NEB PCR Cloning Kit contains a sufficient supply of materials to perform 20 x 10 μl cloning reactions. Primers are also provided, allowing screening for inserts by colony PCR and/or sequencing.
References
Wang, Y. et al. (2004). Nucleic Acids Research. 32, 1197-1207.
Heurgue-Hamard, V. et al. (2000). The EMBO Journal. 19, 2701-2709.
Tenson, T. et al. (1999). Journal of Bacteriology. 181, 1617-1622.
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Specifications & Change Notifications
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The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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