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pSpark® I

For highly Efficient, Accurate & Robust general Cloning from PCR High Fidelity fragments, without toxic genes use

pSpark® I is a highly efficient, accurate and easy-to-use DNA cloning System based on a novel breakthrough technology to generate blunt vectors with a high blunt cloning efficiency.

The vector is prepared by digestion of pSpark® at the EcoRV site before treating both ends to prevent vector self-ligation. The end treatment is supported by a proprietary know-how that guarantees a higher cloning efficiency than with simply a desphorylated vector.

20 rxn

Price: €175.00

SKU: C0001 Categories: ,

Detailed information:

Advantages & Features
  • Unprecedented high cloning efficiency: more than 2,500 positive colonies expected under optimal conditions.
  • Easy-to-use: eliminate recombinant screening due to its minumum background (lower than 1%), avoiding “suicide” strategies from toxic genes.
  • Time-saving protocol: no hidden steps such as phosphorylation, just ligation after PCR and transformation.
  • High stability: eliminates cloning bias or pitfalls.
  • Powerful: clone from less than 1 ng/kb, obtain 5-fold more positive colonies using 10x less DNA insert.
  • Compatible with blue/white screening.
  • Great versatility: compatible with any protocol, proofreading polymerase, competent cells, ligation time or primers.
  • Sensitive: clone from 50 bp insert to up to 14 kb with just 5 ng per kb of insert.
  • Eliminates positive selection vector.
  • High cost-saving: reduces your cloning costs as no expensive phosphorylated primers are needed.
  • Robust for every DNA size: just 6.7 ng per kb of insert needed for optimal ligation.
Specifications

The family of pSpark® DNA cloning vectors
All pSpark® DNA cloning vectors 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 polymerases. Also, 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. Also, all pSpark® DNA cloning vectors have binding sites for pUC/M13 forward and reverse primers and thus the cloned insert can be amplified or sequenced with those primers.
Two versions of the MCS have been developed for pSpark® DNA cloning system: 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. In several pSpark® DNA cloning vectors the MCS has been properly inserted within the alpha-peptide coding region of the enzyme beta-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.
The MCS of the pSpark®-Done Vector contains sequences on either side of the insert that are recognized by the restriction enzymes Not I and EcoR I. This allows the insert DNA to be removed with a single restriction digest using either of these enzymes.
Background of pSpark® vector self-ligation in a ligation reaction without insert produces less than 1% of background of blue colonies when compared with a ligation containing both insert and vector, and thus blue/white screening is not required. For example, under optimal conditions a ligation of 30 ng of a 1 kb insert and transformation into cells with a transformation efficiency of 4×107 cfu/μg produces over 2500 white colonies and less than 20 blue colonies.
Some pSpark® DNA cloning vectors exploit the very low background feature of the vector for the expression of toxic genes under transcription-free conditions. In those variants, the lac promoter has been eliminated and therefore 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 2×107 cfu/μg.
As a help for cloning of inserts with about 8-14 kb, a variant of pSpark® DNA cloning vector with low copy number has been developed. This low copy variant of pSpark® DNA cloning vector is also transcription-free, for the most demanding cloning tasks. Finally, 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.

Includes

Includes for 20 rxn:
– 20 µL pSpark® I (20 ng/µL)
– 20 µL T4 DNA Ligase (5U/Weiss)
– 200 µL T4 DNA Ligase Buffer (5x)
– 150 µL PEG 6000 (10x)
– 5 µL Insert Control 1 kb (20 ng/µL)

Download documentation
Applications
  • General cloning.
  • Cloning of High Fidelity PCR amplified products.
  • Production of ssDNA.
  • Blue/white screening for recombinants.
  • In vitro transcription from T7/SP6 dual-opposed promoters.
Tables & Figures

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Quality Control
  • Functional test using a 1.0 kb PCR fragment.
Advice
  • Recommendations: All pSpark® DNA cloning vectors are stable for at least 1 month at 4 °C and even at 20 – 25 °C for up to 2 days although storage temperatures above -20 °C are not recommended. In case of incident, such as a power failure, stored vectors should be tested with the supplied control insert before considering discarding it. However, please note that T4 DNA Ligase is extremely temperature-sensitive and storage temperatures above       -20 °C inactivates the enzyme.
Storage, Shipping & Guarantee

Shipped in: Gel Pack.
Storage: -20 ºC (NON Frost-Free Freezer).
Shelf Life: 24 months from the date of purchase, if it is properly stored.

Citations
Safety Statements

This product is developed, designed and sold exclusively for Research purposes and in vitro use only (RUO). The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. For more info, please check its Material Safety Data Sheet available in this website.

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