pSpark® DNA Cloning Kits are based on a breakthrough technology to clone without toxic genes, from less than 1 ng/kb, with minimum background (less than 1%) getting an Unprecedented high Efficiency. It have been designed to clone PCR fragments generated with any proofreading DNA Polymerases or Blend DNA Polymerases, and compatible with blue/white Screening. Expensive Phosphorylated Primers are not needed.
All pSpark® DNA Cloning Kits 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 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 Kits have binding sites for pUC/M13 Forward and Reverse Primers and thus cloned insert can be amplified or sequenced with those Primers.
In several 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.
Features & Advantages
- Unprecedented high Cloning Efficiency: get more than 2,500 positive colonies.
- Powerful: clone from less than 1 ng/kb and obtain 5-fold more positive colonies using 10-fold less DNA insert.
- Cost-saving: reduces your cloning costs due no expensive Phosphrylated Primers are needed.
- Easy-to-use: eliminate screening of recombinants due to its minimum background (lower than 1%), without toxic genes.
- Time-saving protocol: no hidden steps such as Phosphorylation, just ligation and transformation.
- High stability: eliminates cloning bias or pitfalls.
- Risk-free: product covered by our Quality 100% Guarantee.
pSpark® Comparison with other popular Systems
In 2016, Canvax™ conducted a rigorous study where the efficiency of all pSpark® Blunt-end DNA Cloning Systems were analyzed in comparison other popular cloning systems, developed almost two decades ago. In this catalog the results of pSpark® I compared to Product P and Product T are presented. If you want to review the full white paper, please press here.
Figure 1.1: Efficiency and background
Figure 1.2: Robust and versatile
Cloning efficiency of pSpark® I over other popular cloning systems. The cells used had a cloning efficiency of 2 x 10⁷ cfu/μg.
Cloning efficiency using pSpark® I with blend polymerase. The 1 kb-insert was amplified with FastPANGEA™ High Fidelity DNA Polymerase MasterMix for cloning with pSpark® I and with blend polymerase to clone with Company P. Competent cells had a cloning efficiency of 2 x 10⁷ cfu/μg.
As shown in the previous figure, the background for pSpark® I is 0.8%, while in other cases, it is 40% and 20%, respectively. On the other hand, pSpark® I has an efficiency of 300 cfu/μg of DNA Insert, while other products have 13 cfu/μg and 17 cfu/μg of DNA, respectively.
Despite the similarity of the results, it is important to highlight that PCR products, obtained with a mix of both DNA polymerases, contain a mixture of molecules with blunt ends and molecules with adenine at the 3´ends in a proportion of 30% and 70%, respectively. Therefore, pSpark® I is more robust and versatile than Product P.
Figure 1.3: Insert amount
Figure 1.4: Ligation Time
Number of positive white colonies obtained after ligation with different ratios of pSpark® I vector:insert. The amount of vector was the same in all cases, varying the amount of insert to achieve the vector: insert ratio identified. The background was less than 1%. Competent cells had an efficiency of 2 x 10⁷ cfu/μg.
pSpark® I ligation-determined efficiency in response to different ligation times. Competent cells used had an efficiency of 2 x 10⁷ cfu/μg. Is possible to use pSpark® using almost any lab protocol, ligation temperature ( example: 25ºC-RT, 22ºC, 16º or 4ºC ), and it could even tolerate some changes depending on the needs of each cloning task or laboratory resources.
As is described, it allows obtaining a high number of colonies even using < 1 ng of insert as in the 5:1 vector: insert ratio.
It is necessary to emphasize that with only 5-10 minutes of ligation time, >400-700 positive colonies and a background <1% are obtained.
Figure 1.5: Insert size
Efficiency of cloning pSpark® I inserts of different sizes using different vector: insert ratios. Inserts were used 0.5 kb, 1kb, 4kb, 7kb and 9kb in the ratios indicated below. Competent cells were 2 x 10⁷ cfu/μg DNA. Background was always below 1%.
As is shown, the vector: insert relationship 1:5 is the best with >2,000 positive colonies for inserts equal or < 1kb.
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Quick links to Products
Blunt-end DNA Cloning Kits
TA DNA Cloning Kits
Universal DNA Cloning Kit*
|pSpark® I||pSpark® TA||pSpark® Universal|
|pSpark® II||pSpark® TA Done|
|pSpark® III||pMBL™ T-Vector||* Compatible with Blunt and TA DNA cloning|
Chemically Competent Cells
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