New England Biolabs, E1202S, NEB® PCR Cloning Kit

Easy cloning of all PCR products, including blunt and TA ends Related Categories DNA Assembly, Cloning and Mutagenesis Kits Specification 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. FAQ Q: How does the NEB® PCR Cloning Kit work? A: Open reading frames coding for 5 amino acid residues or less are difficult for E. coli to process (2,3). Such small genes, or minigenes, lead to premature release of the very small peptide from the ribosome, with the last amino acid residue still covalently attached to its tRNA, such that the tRNA is not readily available for protein synthesis. If the pMiniT 2.0 vector recircularizes without an insert, it will cause lethal inhibition of protein synthesis and no colony will result. If the pMiniT 2.0 vector carries an insert, a colony will grow. Q: How can the cloning vector work with both blunt-ended amplicons and single-base overhang-containing amplicons? A: The mixes contain end-polishing components that will convert single-base overhangs, such as those present in amplicons made by Taq DNA polymerase or Taq-based mixes, to blunt ends. The blunt-end form then can be ligated into the pMiniT 2.0 vector. Q: Do these polishing components present in the master mix affect my cloning efficiency if my insert already has blunt ends? A: No. The concentrations of the end-polishing components are carefully optimized so that any single base overhangs will be removed to form blunt ends; already blunt-ended PCR products are not affected. Q: Do my inserts have to possess 5´ phosphates? A: No, this cloning strategy works well with inserts whether or not they possess 5´ phosphates. In fact, using non-phosphorylated primers for PCR eliminates the possibility of multiple inserts being cloned into the pMiniT 2.0 vector. Q: Can the cloning kit be used for inserts that are not necessarily PCR amplicons? A: Yes, the kit works equally well with blunt-ended and single-base overhang restriction fragments or synthetic DNA. Q: 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? A: Yes, but with lower efficiencies. The polishing components are optimized for removal of a single base overhang. As an extreme test example, cloning a DNA fragment possessing a 4-base, 5´ overhang requiring a fill in and a 5-base, 3´ overhang requiring a chew back resulted in 10-fold fewer transformants than blunt end or single-base overhang DNA fragments. Q: Can I use the NEB PCR Cloning Kit featuring pMiniT 2.0 for Golden Gate Assembly? A: pMiniT 2.0 has no BsaI sites so it can be used to clone Golden Gate Modules that have BsaI sites. Q: Does the PCR product need to be purified? A: No, although higher transformation efficiencies are achieved with purified inserts. If using unpurified PCR amplicons, use 1 μl or less of the PCR as insert material to achieve a 3:1 molar ratio of insert:vector. It is especially important for high yield PCR situations to avoid adding a vast excess of insert that would result in the insert ligating to both ends of the vector backbone. Q: Can I use a different competent E. coli strain than the provided NEB 10-beta strain? A: While this cloning system works well with a variety of cell strains, it is important to use a cell strain with robust growth to maintain selection pressure towards plasmid constructs containing your insert. In addition to the provided NEB 10-beta Competent E. coli (Cloning Efficiency) in NEB PCR Cloning Kit (NEB #E1202), cell strains that work well with this cloning kit include NEB Turbo (NEB #C2984) and NEBExpress (NEB #C2523) Competent E. coli. The 5-alpha strains are not recommended, as their slightly slower growth rate does not support strong background suppression. Q: How can I maximize the number of transformants? A: Use the longer suggested time periods for the protocol steps. If desired, plate additional 50 μl aliquots of the 1 ml outgrowth onto additional plates, but do not plate more than 50 μl per plate to maintain a low background. Q: Can I scale down the reactions to use less vector? A: The vector used per reaction can be decreased from 25 ng to 10 ng. Decrease the amount of insert proportionally to maintain the optimal 3:1 insert:vector molar ratio, and also the amount of added Cloning Mix 1 and Cloning Mix 2. Q: Are there limits regarding the size of inserts that can be cloned? A: NEB 10-beta supports large plasmid stability, and the small size of pMiniT allows large insert cloning. Inserts of 4–5 kb have been easily cloned. Note that larger inserts may require lower insert-to-vector ratios. Q: 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)? A: The cells are the same, but differ 10-fold in their transformation efficiencies. Our testing indicates the supplied cloning efficiency level supports excellent results for PCR amplicon cloning. Q: How can I determine if my NEB 10-beta cells are competent? A: The pUC19 control DNA included in the kit is provided in case the user would like to confirm the competency of the provided NEB 10-beta Competent E. coli cells. While this is not routinely required, the transformation efficiency can be independently confirmed by transforming 100 pg (2 μl of the 50 pg/μl stock) into 50 μl competent cells and following the normal transformation and plating protocols. The resulting transformation efficiency should be > 1 x 108 cfu/μg pUC19 DNA. Q: Can Cloning Mix 1 and Cloning Mix 2 be mixed together before adding them to the ligation reaction? A: Cloning Mix 1 and 2 can be mixed before setting up the reaction. For long term storage Cloning Mix 1 and Cloning Mix 2 should be stored in their separate vials. Storing mixed Cloning Mix 1 and 2 together will result in reduced enzyme activity. Q: Where are the +1 transcription positions for the SP6 and T7 promoters for in vitro transcription and translation located? A: The +1 position for the SP6 promoter is located 50 base pairs upstream from the cloning site for transcription of inserts cloned in a CW orientation, while the +1 position for the T7 promoter is located 62 base pairs downstream from the cloning site for transcription of inserts cloned in a CCW orientation. Q: What is the difference between the original pMiniT and the pMiniT 2.0 linearized vector backbone now provided in the kit? A: The 2.0 version features three improvements: Addition of T7 and SP6 RNA polymerase promoter sequences flanking the cloning site for in vitro transcription of cloned inserts. Addition of seven new restriction sites including four 8 base recognition restriction enzyme sites flanking the cloning site to allow easier linearization downstream of the cloned insert for transcription studies, and to increase options for subcloning strategies Elimination of the BsaI site present in the ApR gene through site-directed mutagenesis to allow easier downstream use of cloned inserts/modules for Golden Gate Assembly. Both versions of the linearized vector backbone have identical functionality in the cloning kit. Q: When cloning a PCR fragment into the pMiniT 2.0 vector provided with the NEB PCR cloning kit, where in the multiple cloning site (MCS) of the vector is the insert ligated? A: The insert will be ligated between nucleotides 545 and 546 of the pMiniT 2.0 vector. This insertion point is located between the recognition sites for EcoRi and PacI in the MCS region of the vector.