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. To learn more about how the kit works, please view our video.
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:
NEB® 10-beta Competent E. coli (Cloning Efficiency)
C3018AVIAL
-80
20 x 0.05 ml
Not Applicable
Properties & Usage
Storage Notes
The kit is shipped on dry ice. Upon arrival, store the competent cells (in the large
exterior box) at -80°C, the components in the small interior box at -20°C and the
NEB™ 10-beta/Stable Outgrowth Medium at room temperature or 4°C.
Advantages and Features
Features
In vitro transcription with both SP6 and T7 promoters
BsaI-site removed from the ampicillin-resistant gene (allows for the cloning of Golden Gate Assembly modules)
Easy cloning of all PCR products, including blunt and TA ends
Fast cloning experiments with 5-minute ligation step
Simplified screening with low/no colony background and no blue/white selection required
Save time by eliminating purification steps
Flanking restriction sites available for easy subcloning, including choice of two single digest options
Provided analysis primers allow for downstream colony PCR screening or sequencing
Ready-to-use kit components include 1 kb control amplicon, linearized cloning vector and single-use competent E. coli
Longer shelf life (12 months), as compared to some commercially available products
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.
1. Use an insert:vector ratio of 3:1: A higher insert:vector ratio can actually result in fewer colonies. This is due to the fact that inserts may ligate to both ends of the vector, which will prevent the cloning of your amplicon insert.
2. Follow the protocol: The protocol has been highly optimized to have a low background; if you have inadvertently deviated from the optimized protocol (e.g., extended ligation incubation, overly-concentrated outgrowth), compensate by plating less outgrowth (< 50 µl) - Plating too much of the outgrowth can increase background, and cause problems with colony PCR. If you need more colonies, spread 50 µl of outgrowth onto each of multiple plates.
3. Important to stop ligations: If you wish to store your ligations to allow transformations at a later time, make sure your freezer is cold enough (- 20°C) to freeze the ligations. Or, you may quick freeze with a dry ice/alcohol bath before transferring the samples to -20°C. If you find your freezer-stored ligations have remained in liquid form, this may have allowed further low-level ligation of the vector backbone to occur. In this circumstance, plate less outgrowth (< 50 µl).
4. Do not incubate the transformation plates at room temperature. The slow growth rate of the cells at room temperature will increase the number of background colonies.
5. Add the cloning mixes 1 and 2 to the reaction last. Some people try to save time by preparing a mix of water, ligation master mix and pMiniT, aliquoting this to tubes and adding the insert DNA. This allows pMiniT to recircularize, since ligation can begin before the amplicon is added, and this may result in lower cloning efficiency.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
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]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Trademarks
ONETAQ®, Q5®, QUICK-LOAD®, NEB® and NEW ENGLAND BIOLABS® are registered trademarks of New England Biolabs, Inc. LONGAMP™ and PMINIT™ are trademarks of New England Biolabs, Inc.
PHUSION® is a registered trademark and property of Thermo Fisher Scientific. Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific.
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