pSpark®
Probably the best DNA cloning technology ever developed
- Blunt DNA cloning with less than 1% background
- Not a troublesome positive selection vector
- Cloning possible with 0,1 ng of non-optimized PCR
pSpark® and your time
pSpark® DNA cloning systems in combination with the recent development of highly robust high fidelity DNA polymerases makes DNA cloning an extremely easy task. pSpark® system saves your valuable time due
High efficiency of pSpark® systems.
Due to the extremely high cloning efficiency of pSpark® DNA cloning systems, ligations can be performed
When only 10 minutes of ligation are used the number of colonies obtained is about the same than a T/A cloning reaction made in parallel under optimal conditions (60 minutes to overnight ligation at 16ºC).
Generally, incubation at 22ºC for 60 minutes produces about 5 times more transformants than a ligation for 10 minutes.
For example, using 7 ng of the supplied Control Insert for a ligation in 10 minutes, a fast transformation protocol and competent cells with a transformation efficiency of 4x107 cfu/µg about 400 to 500 white colonies are obtained. About 900 to 1000 white colonies are obtained when ligation time is increased to 30 minutes.
Overnight incubation does not improve nor is detrimental for transformation.
Use of PCR product directly for cloning
PCR amplified DNA should be analysed on an agarose gel before use in the ligation reaction to verify both the quality and quantity of your PCR product. A PCR that contains one single homogeneus band with only primers dimers can be purified by any high quality PCR clean-up kit for cloning into pSpark® DNA cloning systems.
In several cases crude unpurified PCR products can be used directly for ligation into pSpark® DNA cloning systems but even in those cases we recommend to run an aliquot of PCR amplified DNA by gel electrophoresis to check both quality and quantity.
Use a fast protocol of transformation
An alternative transformation protocol of only 5 minutes could be used to transform with pSpark® ligation reactions.
The main difference between this protocol and the standard protocol is that this fast protocol avoids the heat shock step and instead this essential step occurs directly on the plate. If you feel unfamiliar with this protocol, please use the standard transformation protocol.
No sequence mistakes
One major drawback of Taq is its relatively low replication fidelity. Due to the fact that error rate estimation varies among different assays employed and by different labs performed it, this feature is one of the most discussed characteristics of thermostable polymerases.
In any way, it is undiscuss that the error rate of Taq polymerase is superior to that of proofreading polymerases. Error rate of Taq polymerase has been reported to be 1 mistake for every 1 x 104 to 9 x 105 bases polymerized, first generation of high fidelity polymerases (e.g. Pfu) introduce 1 wrong base for every 1.5 x 106 bases polymerized and second generation of high fidelity polymerases introduce a wrong base for about every 4 x 107 bases polymerized.
What is pSpark?
pSpark® is a novel blunt DNA cloning vector with very high cloning efficiency. It is not a dephosphorylated vector nor a troublesome positive selection selection vector and thus DNA cloning is extremely easy. Click on the images or below to know more.
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News
Canvax Biotech SL will be at the XXXIII Congress of the Spanish Society of Biochemistry and Molecular Biology (SEBBM) at September 15-17th. We will participate with a scientific poster so you can visit us in these area.
Our poster is about the comparison of pSpark® DNA Cloning System with the methods of cloning presents in the market. We will be pleased to meet you in Cordoba and to answer all your questions related to novel technologies for DNA cloning, laboratory protocols ...