Long-fragment cloning
| Long fragment cloning is often challenging due to the limitations of traditional molecular biology reagents. Invitrogen has developed novel cloning products that overcome these limitations and enable precise and efficient assembly of long fragments. |
Seamlessly Clone Fragments up to 10 kb
) | GeneArt® Seamless Cloning and Assembly enables cloning of DNA fragments up to 10 kb in any vector in 30 minutes.This figure outlines the GeneArt® Seamless Cloning and Assembly protocol for assembling up to four DNA fragments and a vector simultaneously and in precise order.
|
Seamlessly Clone Fragments over 10 kb
) | GeneArt® High-Order Genetic Assembly enables simultaneous and seamless assembly of existing DNA fragments up to 100 kb.
|
TOPO® Clone Fragments up to 10 kb
The TOPO® XL PCR Cloning Kit combines TOPO® cloning, Zero Background™ technology, and a unique gel purification step to enhance cloning of PCR products from 3 to 10 kb. Convenient features of the pCR®-XL-TOPO® vector include:
- ccdB gene eliminates background of non-recombinant clones
- Kanamycin and Zeocin™ resistance genes for your choice of selection in E. coli
- T7 sequence for in vitro RNA transcription and sequencing
- Selection of competent cells for high speed, electroporation, or routine cloning
| Unique gel purification step improves results Long PCR often yields multiple products, making gel purification necessary prior to cloning. However, gel purification requires exposure to ethidium bromide and UV light, which can nick and damage DNA. To protect against nicking, the TOPO® XL PCR Cloning Kit uses crystal violet to enable visualization of DNA bands in an agarose gel under ambient light. This eliminates the need for ethidium bromide and UV light exposure, ensuring safe gel purification. Crystal violet staining results in significantly more colonies and a greater percentage of recombinants than using ethidium bromide and UV light (Table 1). Figure 3: pCR®-XL-TOPO® vector. |  |
Table 1 - Crystal violet enhances TOPO® cloning of large fragments.
A 7 kb ampicilin resistance gene sequence was PCR-amplified, and PCR products were loaded onto one gel with crystal violet and another gel stained with ethidium bromide. PCR products were gel purified and cloned into the pCR-XL-TOPO® vector. The number of recombinants was determined by plating 125 of each transformation on LB plates containing either kanamycin or kanamycin and amplicillin.
| Crystal violet | Ethidium bromide | |
|---|---|---|
| Total colonies (Kan2) | 275 | 15 |
| Colonies w/Insert (Kan2-Amp2) | 258 | 9 |
| Parent recombinants | 94% | 60% |
