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
Selecting the right pSpark®
pSpark® DNA cloning system allows some useful applications such as
- Highly efficient DNA cloning of PCR products amplified with proofreading DNA polymerases
In vitro transcription from T7/SP6 dual-opposed promotersCloning of toxic genes (ONLY transcription-free vector variants)- Cloning of unstable genes, for example genes with repetitive sequences (ONLY transcription-free and low copy vector variants)
Production of ssDNA as a filamentous phage f1 Blue/White screening for recombinants (NOT available in transcription-free variants)Cloning directly from PCR using plasmid cloned genes as template (particularly vectors with dual antibiotic resistance to avoid background due to template)
Available variants of pSpark® DNA cloning kits and features of each one are listed below
| vector | OriC ( copy number ) |
MCS | Blue/White feature | Antibiotic | Size ( bp ) | Application |
| pSpark® I | pUC ( High ) | Advanced | Yes | Amp | 3013 | General Cloning |
| pSpark® II | pUC ( High ) | Classical | Yes | Amp | 3001 | General Cloning |
| pSpark® III | pUC ( High ) | Advanced | Yes | Amp / Kan | 3980 | Unpurified PCR Cloning |
| pSpark® IV | pUC ( High ) | Advanced | No transcription-free |
Amp | 3369 | Toxic genes cloning |
| pSpark® V | pUC ( High ) | Advanced | Yes transcription-free |
Amp | 3013 | Unstable and toxic genes cloning |
Two versions of the MCS have been developed for pSpark® DNA cloning systems: one classic MCS (cMCS) with only one nucleotide difference from the popular MCS derived from pGEM® vector from Promega and one advanced MCS (aMCS) with blunt restriction enzymes at each side of the cloned insert, 8bp rare cutters at each side of cloned insert, inexpensive restriction enzymes recognition sites at each side of cloned insert, enzymes that generate ends compatible each other at each side of cloned insert and enzymes with activity in several buffers for fast and inexpensive analysis of recombinants.
Also, all pSpark® DNA cloning systems have binding sites for pUC/M13 Forward and Reverse primers and thus cloned insert can be amplified or sequenced with those primers.
1
In vitro transcription from dual-opposed promoters
All pSpark® DNA cloning systems comprise vectors that contain both T7 and SP6 RNA polymerase promoters flanking the multiple cloning region (MCS) for in vitro transcription of cloned DNA using either T7 or SP6 RNA polymerises. In vitro transcription from dual-opposed promoters permits cloning of toxic genes or cloning of unstable genes (repeated sequences).
3
Cloning of toxic genes
Some pSpark® DNA cloning vectors exploit the very low background feature of the vector for the expression of toxic genes under transcription-free conditions. Those vectors lacks of lac promoter thus blue/white selection is not possible. This feature allows the cloning of DNA that when transcribed and translated produce toxic proteins.
4
Production of ssDNA as a filamentous phage f1
All vectors belonging to pSpark® DNA cloning vectors family have the origin of replication of the filamentous phage f1. Synthesis of single-stranded DNA requires phage encoded gene II, X and V and is initiated at f1 ori.
2
Blue/white screening for recombinants
In several pSpark® DNA cloning vectors the MCS has been properly inserted within the alpha-peptide coding region of the enzyme β-galactosidase for insertional inactivation of the alpha-peptide by recombinant clones thus allowing positive clones to be directly identified by Blue/White screening on X-Gal plates. In some variants of pSpark®, the lac promoter has been eliminated and thus Blue/White screening is not allowed but those vectors are useful for cloning genes that produce toxic polypeptides by transcription/translation. For those transcription-free pSpark® DNA cloning vectors a background of less than 1% white colonies should be expected under optimal conditions. No more than 30 white colonies are obtained in a ligation without insert when using cells with a transformation efficiency of 4x107 cfu/µg.
5
Cloning directly from PCR using plasmid cloned genes as template
There are variants of pSpark® DNA cloning vector with either ampicillin resistance or with combined ampicillin and kanamycin resistance. The vector with both ampicillin and kanamycin resistance is useful for cloning PCR products amplified from any plasmid vector without the need to gel purify bands to eliminate the background due to the template vector used for PCR.
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 ...
Get a free sample of pSpark® DNA Cloning Vector in the web site www.psparkcloning.com/web/index/samplerequest. Test it and send us your lab result before May 10, 2010th. So, you will enter in a raffle of a iPod nano 8Gb RED on May 12, 2010th.
We think that pSpark® DNA Cloning System are, probably, the best cloning system in the market. So we wish you that you test it and discover your adventajes: efficiency, low or not background, easy to use, low amount DNA cloning, you must to ...