The pSTBlue-1 Perfectly Blunt Cloning Kit is designed for simplified cloning of PCR products amplified with any DNA polymerase. The kit contains a special End Conversion Mix, T4 DNA Ligase, pSTBlue-1 Blunt Vector, NovaBlue Singles Competent Cells, SOC Medium, Blunt Vector Control Insert and Test Plasmid. DNA fragments can be PCR amplified with any DNA polymerase, including error-proof polymerases that give a lower yield of the PCR product, but guarantee high-fidelity performance. No PCR purification is necessary if a strong discreet band is visible on the agarose gel electrophoresis. The ends of the PCR product are converted to a blunt-phosphorylated form in the end-conversion reaction for 15 min at the room temperature. Subsequently, the insert is directly ligated to the pSTBlue-1 blunt vector in an optimized short ligation reaction with T4 DNA Ligase. The ligation reaction is immediately used to transform pre-packaged NovaBlue Singles Competent Cells according to the enclosed protocol. To determine transformation efficiency, one of the NovaBlue Singles is transformed with provided Test Plasmid. Cells are plated on the LB agar plates containing IPTG, X-gal and carbenicillin. The plates are incubated overnight at 37C and the blue/white recombinants are identified next day. It is expected that white colonies contain vector + insert and blue colonies contain vector only. To confirm insert presence, colony PCR is routinely performed. After positive clones are identified, high-copy plasmids containing inserts are isolated and sequenced.
The pSTBlue-1 Perfectly Blunt Cloning Kit can be used for the purpose of general cloning, archiving, subcloning, sequencing and in vitro transcription. The pSTBlue-1 vector contains dual opposed SP6/T7 promoters for in vitro transcription and sequencing, dual EcoRI sites flanking insert and provides either ampicillin (carbenicillin) or kanamycin selection.
We selected the pSTBlue-1 Perfectly Blunt Cloning Kit to perform cloning of the gene coding for a functional RNA molecule. The PCR product was amplified using AccuPrime Taq DNA Polymerase (Invitrogen). The cloning procedure was very easy to perform. We identified a number of positive white colonies, in addition to the negative blue ones. All the positive and negative controls worked as expected. To confirm insert presence, white colonies were picked using a pipet tip and directly amplified using colony PCR. Interestingly, in addition to blue and white colonies, we have encountered a number of light blue colonies. When we analyzed them by colony PCR we found that more than 75% of them contained the inserts of the expected size. After growing an overnight bacterial culture from the positive clones, the plasmid was inserted and the sequence was confirmed by cycle sequencing. The sequencing results generally confirmed the presence of the correct insert. Occasionally, only vector sequence was identified through sequencing indicating that the insert was lost.
Presence of the dual SP6 and T7 promoters in the pSTBlue-1 vector was a very important feature. As with any blunt cloning kit, the insert was frequently ligated in the opposite orientation. We were able to circumvent this problem by using either SP6 or T7 promoter for in vitro transcription, depending on the orientation of the insert.
In summary, we feel that we had a great success with pSTBlue-1 Perfectly Blunt Cloning Kit. The reaction needed no optimization or troubleshooting. The result was satisfactory and everything worked well the first time.
Yelena Bykhovskaya PhD
Research Scientist I
Molecular Hematology Laboratory
Cedars-Sinai Medical Center Los Angeles CA