Kit includes everything necessary to clone and select recombinant vectors containing your PCR fragment of choice. We have removed much of the multiple cloning site from the pCR4Blunt-TOPO vector to shorten the distance between sequencing primer sites and the insert site to as little as 33 bp. This means sequencing reactions give less vector sequence and more insert sequence. The pCR4Blunt TOPO vector has sites for four common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.
pCR4Blunt-TOPO Clone Selection and Manipulation
The pCR4Blunt-TOPO vector contains both ampicillin and kanamycin resistance markers and a LacZα-ccdB gene fusion for positive selection and blue/white screening. The vector's minimized multiple cloning site still includes flanking EcoRI sites for simplified excision of cloned PCR products and a unique Sse8387I site for generation of nested deletions prior to sequencing. T7 and T3 promoters are also present for in vitro transcription.
Simplified TOPO-Based Cloning
Using TOPO cloning technology there is no need for PCR primers containing specific sequences, post-PCR procedures, vector preparation, or other time-intensive DNA manipulation steps. Just add your PCR reaction straight to the provided topoisomerase-charged vector, incubate 5 minutes, and transform the provided E. coli competent cells.
With up to 95% of clones carrying the desired insert, you can screen fewer clones to help you save time and money. The pCR4Blunt-TOPO vector used in this kit comes with no overhangs for efficient ligation of PCR products created by proofreading, thermostable polymerases that leave blunt-ended PCR products.
The Standard in Cloning
When it comes to cloning, TOPO cloning technology has been a reliable partner for thousands of scientists for over ten years. Fast, simple-to-use, and efficient, TOPO cloning has been applied to many different vectors for a wide array of applications.
Zero Blunt TOPO PCR Cloning Kits for Sequencing Kit Options
The Zero Blunt TOPO PCR Cloning Kit for Sequencing can be purchased with a variety of competent cells that deliver different advantages depending on what your needs are:
- General cloning: TOP10 (Cat. Nos. K2875-J10, K2875-20, K2875-40)
- High efficiency cloning: TOP10 Electrocomp™ Cells (Cat. Nos. K2880-20, K2880-40)
- General cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. No. K2895-20)
- Fast growth: Mach1™-T1R Chemically Competent E. Coli (Cat. No. K2835-20)
- Get More Sequence: Allows for more insert sequence and less vector sequence when using standard sequencing primers
- Fast and Easy: Go from PCR-to-clones in just three steps and in as little as 5 minutes hands-on time
- Efficient: Achieve up to 95% clones with correct insert
- Proven: Reliable performance for over a decade with over 4000 citations
- VECTOR: pCR4Blunt-TOPO Vector: Optimized cloning vector for improved sequencing results
- CLONING METHOD: TOPO Cloning: Topoisomerase I based 5 minute ligation of proofreading-polymerase-amplified PCR products with blunt ends to the vector
- COMPETENT CELLS: Various Options: Choose from kits with either general, high-efficiency, bacteriophage T1-resistant, or fast-growing competent cells
- pCR4Blunt-TOPO Vector: Optimized for Sequencing
Chromatin Biology, Chromatin Immunoprecipitation (ChIP), Cloning, Methylation Analysis, PCR Cloning, RNAi, Epigenetics and Non-Coding RNA Research, Sequencing Bisulfite-Treated DNA
|Kit, PCR Cloning for Sequencing|
|Chromatin Biology, Chromatin Immunoprecipitation (ChIP), Cloning, Methylation Analysis, PCR Cloning, RNAi, Epigenetics and Non-Coding RNA Research, Sequencing Bisulfite-Treated DNA|
|Box 1: Topoisomerase I-activated pCR4Blunt-TOPO vector, Salt solution, dNTPs, Control template, T3, T7, M13 forward, and M13 reverse primers, Control PCR primers, Sterile water. Box 2: One Shot Chemically Competent or Electrocomp E. coli, S.O.C. medium, Supercoiled pUC19 control plasmid|