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 inflexibility:clone with any amplicon made with anyDNA polymerase, with or without 5´ phosphates, purified or not!PCR cloning withlow/no backgroundA 500 bp PCR product incubated with the linearized vector in a 3:1 ratioaccording to recommended protocol. 2 μl of reaction was transformed intoprovided NEB 10-beta Competent E. coli and 1/20th of the outgrowth wasplated. Left plate serves as the control, with vector backbone only, right platecontains PCR insert.Cloning Kit Protocol OverviewpMiniT 2.0 Vector MapMap shown above displays the construct formed if no insert is present. Unique restriction sites are shownin black. Additional restriction sites that can be used for subcloning are shown in red. Expanded box belowshows location of sequencing primers, restriction sites for subcloning, and placement of insertion sitewithin the toxic minigene.
This product is related to the following categories:
DNA Assembly, Cloning and Mutagenesis Kits Products
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