 | | | | USER Friendly Cloning Kit |
| | | | |
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
- Fast, simple and extremely efficient method for cloning PCR products
- >95% cloning efficiency within a wide range of PCR product concentration
- Simply mix PCR product with USER Enzyme and vector; incubate for 30 minutes; transform
- No PCR product clean-up required
- Choice of insert orientation
- Can be used with any strain of competent cells
- Any vector can be adapted to be "USER Friendly" with the Universal USER Cassette
Description:
The USER™ (uracil-specific excision reagent) Friendly Cloning Kit offers an extremely fast, easy and efficient method for the cloning of PCR products (1). This novel method is not dependent on restriction enzyme cleavage, nor does it require DNA ligase for insertion of PCR product into vector. Instead, vector-specific PCR primers which contain one uracil per primer are designed and the target DNA is amplified with Taq DNA Polymerase; next, the resulting PCR products are treated with the USER™ Enzyme to create unique 3´ single-stranded extensions which can then anneal to the supplied linearized vector (Figure 1).
Linearized plasmid vector, pNEB206A, which has an 8-nucleotide, 3´ single-stranded extension on both ends (1), is supplied with the kit. The single-stranded extensions are not complementary; this prevents the vector termini from re-annealing to form transformable circular DNA and also allows control over the orientation of the inserted PCR product.
To provide vector-compatible extensions on PCR-amplified fragments, primers should be designed with 8 additional nucleotides at their 5´ ends (Figure 1A). These 8 nucleotides are identical to the single-stranded extensions on the vector, except for the 3´ thymine, which in the primer sequences is replaced by a deoxyuridine (dU). Downstream of these 8 nucleotides, the primer sequences are complementary to the target DNA sequence. In the presence of natural dNTPs, Taq DNA Polymerase incorporates adenine opposite the template-strand uracil. After amplification, each end of the target DNA is extended by 8 nucleotides with a single uracil residue at the junction (Figure 1B). Next, the uracil residues are excised from the amplified product using the USER™ Enzyme (1). USER Enzyme specifically nicks the uracil-containing strand of the PCR product, generating a single nucleotide gap at the location of the nick. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endo VIII. UDG catalyses the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact (2,3). The lyase activity of Endo VIII breaks the phosphodiester backbone at the 3´ and 5´ sides of the abasic site so that base-free deoxyribose is released (4,5). After phosphodiester bond breakage, the terminal 7-mer oligonucleotides dissociate, leaving the PCR fragment flanked by 8-nucleotide long 3´ single-stranded extensions. When mixed together, the vector and USER-treated PCR fragment assemble into a recombinant molecule, as their 3´ single-stranded extensions are complementary to each other (Figure 1B). Because the extensions are so long, ligation is not required. The construct is now ready for transformation of chemically competent E. coli cells.
On average, 104 desired recombinants can be obtained per reaction (when the cell competency is 5 x 106 c.f.u./µg DNA) and greater than 95% cloning efficiency is routinely achieved within a wide range of PCR product concentration.
Figure 1: (A) Design strategy of the pNEB206A vector compatible primers. An optional ATG should be included if protein expression is desired. (B) PCR product cloning method using the USER Friendly Cloning
Figure 2: Effect of PCR product concentration on the cloning efficiency.
Advantages:- 30 minutes from completed PCR to transformation
Kit Components:
M13/pUC Reverse Sequencing Primer (-48) (24-mer)
M13/pUC Sequencing Primer (-47) (24-mer)
pNEB206A Linearized Vector
USER Enzyme
Storage Conditions
Storage Temperature:
-20°C
References
- Bitinaite, J., New England Biolabs, unpublished observations.
- Lindahl, T., Ljungquist, S., Siegert, W., Nyberg, B. and Sperens, B. (1997) J. Biol. Chem., 252, 3286-3294.
- Lindhal, T. (1982) Annu. Rev. Biochem., 51, 61-64.
- Melamede, R.J., Hatahet, Z., Kow, Y.W., Ide, H. and Wallace, S.S. (1994) Biochemistry, 33, 1255-1264.
- Jiang, D., Hatahet, Z., Melamede, R.J., Kow, Y.W. and Wallace, S.S. (1997) J. Biol. Chem., 272, 32230-32239.
Reagents Sold Separately
M13/pUC Reverse Sequencing Primer (-48) (24-mer)
M13/pUC Sequencing Primer (-47) (24-mer)
pNEB206A Linearized Vector
USER Enzyme
Companion Products
LITMUS™ U
Universal USER Cassette
|
