pSpark®

Q1. What are the pSpark® DNA cloning systems?

A1. The pSpark DNA cloning systems are a family of cloning vectors for blunt end DNA cloning. They are based on a novel patent pending technology for background reduction. Vectors are digested with EcoRV and both ends are treated to reduce background to less than 1% . They are not T-vectors. Also, they are not based on transcription of toxic genes to reduce background and for this reason, ORF and promoters can also be cloned into pSpark® DNA cloning systems without cloning bias or artifacts.

Q3. I have used an enzyme blend containing Taq DNA Pol and a proofreading DNA Pol (e.g. Expand™ High Fidelity PCR System, a Taq/Tgo blend) for amplification. Should I clone this PCR product into pSpark® DNA cloning systems? Do I need any additional step (e.g. a blunting step)? Which are the expected results?

A3. Yes. pSpark® DNA cloning systems could be used for cloning PCR products amplified with enzyme blends without any additional step such as a blunting step. In fact, when such inserts were cloned in parallel into a T/A based vector and into pSpark® DNA cloning systems a slightly higher number of colonies were obtained with pSpark® DNA cloning systems using 10 times less DNA. No additional blunting step is needed because such amplified DNA is a mix of both 3´-Adenine overhanging and blunt ended DNA molecules.

Q5. Do I need to purchase phosphorylated primers or to phosphorylate PCR product for cloning into pSpark® DNA cloning systems? Do primers need any tail at their 5´-end for cloning into pSpark® DNA cloning systems?

A5. No. Any primer you already have in your lab can be used for cloning into pSpark® DNA cloning systems. Phosphorylated primers do not inhibit ligation and thus can also be used. There is no need to add any specific sequence at 5´-ends of primers for cloning into pSpark® DNA cloning systems.

Q7. Do I need to change any protocol, E. coli strain or media I am using now to change to pSpark® DNA cloning systems?

A7. No. Just set up a ligation according to catalogue Section 2.3.1 (or Quick Protocol for Advanced Users), incubate ligation up to 60 minutes at 22ºC (or overnight if needed) and follow all the regular protocols you are familiar with. Use a 3:1 to 5:1 (recommended) insert to vector molar ratio as starting point. That corresponds exactly to 6,7 ng of insert per kb for the recommended 5:1 insert-to-vector ratio. As a rule of thumb you can use 5ng per kb of insert to be cloned, that is, 2,5 ng, 5 ng or 10 ng if cloning an insert of 0,5 kb, 1kb or 2 kb. If cloning an unpurified PCR product use 1 uL of PCR directly for ligation.

Q9.Many suppliers and users do not recommend the fast transformation protocol (Section 2.4.3) to be used to transform ligations. Why do you recommend this protocol for cloning into pSpark® DNA cloning systems?

A9. pSpark® DNA cloning systems are by far the most efficient cloning kits developed to date and in fact this supplied fast transformation protocol has been developed and tested to be used with pSpark® DNA cloning systems. Using this fast protocol of only 5 minutes transformation, cells with a transformation efficiency of 4x107 cfu/µg and the supplied control insert, about 500-1000 positive white colonies are obtained instead of over 2500 positve colonies when using the standard protocol.

If you feel uncomfortable with this protocol, use the standard protocol but as a suggestion to save your precious time we recommend to test this protocol. We do not know, however, if for example, method to prepare competent cells, E. coli strain used, temperature inside air flow cabinet, transformation protocol used or plating method have influence in outcome. To date we have no customer who have tested this fast transformation protocol for cloning into pSpark® with unsatisfactory results. If you test this protocol in parallel with the standard transformation protocol with unsatisfactory results please contact us to know more about your experience and to make it available to the rest of the scientific community.

Q11.Do pSpark® DNA cloning vectors are mixed or bound to any protein?

A11. No. Vector vial has only a highly stable DNA vector and this contributes to the very high stability of pSpark® DNA cloning vectors that could be even shipped at room temperature (only samples and kits without ligase). We have stored freeze-dried pSpark® DNA cloning vectors up to 30 days at 20-24ºC with no loss in cloning efficiency. However, we recommend storage of vector at -20ºC.