NEB® PCR Cloning Kit


  • For both Blunt and T/A Cloning
  • 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. 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 background
A 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 OverviewProtocol

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.

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
Cloning Mix 1-202.5X
Cloning Mix 2-2010X
Linearized pMiniT 2.0 Vector-2025 μg/ml
Amplicon Cloning Control-2015 ng/μl
Cloning Analysis Forward Primer-20100 μM
Cloning Analysis Reverse Primer-20100 μM
NEB 10-beta Competent E. coli (Cloning Efficiency)-80
pUC19 Vector-200.05 ng/μl
NEB® 10-beta/Stable Outgrowth Medium4

Advantages and 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

Properties and 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.


  1. 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.


  1. Wang, Y. et al. (2004). Nucleic Acids Research. 32, 1197-1207.
  2. Heurgue-Hamard, V. et al. (2000). The EMBO Journal. 19, 2701-2709.
  3. Tenson, T. et al. (1999). Journal of Bacteriology. 181, 1617-1622.


  1. How does the NEB® PCR Cloning Kit work?
  2. How can the cloning vector work with both blunt-ended amplicons and single-base overhang-containing amplicons?
  3. Do these polishing components present in the master mix affect my cloning efficiency if my insert already has blunt ends?
  4. Do my inserts have to possess 5´ phosphates?
  5. Can the cloning kit be used for inserts that are not necessarily PCR amplicons?
  6. Can the cloning kit be used with inserts containing 5´ or 3´ overhangs greater than the single-base overhang achieved by PCR with Taq DNA polymerase?
  7. Can I use the NEB PCR Cloning Kit featuring pMiniT 2.0 for Golden Gate Assembly?
  8. Does the PCR product need to be purified?
  9. Can I use a different competent E. coli strain than the provided NEB 10-beta strain?
  10. How can I maximize the number of transformants?
  11. Can I scale down the reactions to use less vector?
  12. Are there limits regarding the size of inserts that can be cloned?
  13. Are the NEB 10-beta Competent E. coli (Cloning Efficiency) provided in the kit the same cells as NEB 10-beta Competent E. coli (High Efficiency)?
  14. How can I determine if my NEB 10-beta cells are competent?
  15. Can Cloning Mix 1 and Cloning Mix 2 be mixed together before adding them to the ligation reaction?
  16. Where are the +1 transcription positions for the SP6 and T7 promoters for in vitro transcription and translation located?
  17. What is the difference between the original pMiniT and the pMiniT 2.0 linearized vector backbone now provided in the kit?

Tech Tips

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.


  1. Ligation Protocol for NEB PCR Cloning Kit
  2. Transformation Protocol for NEB PCR Cloning Kit
  3. Plating Protocol for NEB PCR Cloning Kit
  4. Insert Screening Protocols for NEB PCR Cloning Kit
  5. RNA Synthesis of Cloned Insert Transcripts


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Usage Guidelines & Tips

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.