NEB® PCR Cloning Kit
Now supplied with outgrowth medium optimized for NEB 10-beta & NEB Stable Competent E.coli ; please do not use SOC outgrowth medium.
Easy cloning of all PCR products, including blunt and TA ends
- 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)
- 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
- Updated to allow for in vitro transcription with both SP6 and T7 promoters
- Includes competent cells
- More restriction sites added, including four 8-base cutters
How Does the NEB PCR Cloning Kit Work?
NEB TV Ep. 8 – Cloning and DNA Assembly
Overview of PCR Cloning
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!
The following reagents are supplied with this product:
Store at (°C) Concentration NEB® 10-beta/Stable Outgrowth Medium 4 1 X NEB® 10-beta Competent E. coli (Cloning Efficiency) -80 Cloning Mix 1 -20 2.5 X Cloning Mix 2 -20 10 X Linearized pMiniT™ 2.0 Vector -20 25 μg/ml Amplicon Cloning Control -20 15 ng/μl pUC19 Vector -20 0.05 ng/μl Cloning Analysis Forward Primer -20 100 μM Cloning Analysis Reverse Primer -20 100 μM
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