Cloning Without Ligase:
A Fast, Accurate Alternative
Data kindly provided by:
Study 1: Ti-yu Lin, Graduate Student, Univ. of Wisconsin–Madison
Study 2: Madhusudan Rajendran, Research Assistant, Univ. of Wisconsin–Madison
In these studies, In-Fusion Cloning was used for cloning experiments that had been previously unsuccessful with traditional ligation-based methods. Both experiments utilized pET vectors, a commonly used system for in vivo expression of recombinant proteins. When using ligase, both researchers were unable to clone their desired fragments—Ti-yu Lin working with a ~1-kb GC-rich insert (67% GC content), and Madhusudan Rajendran working with a large insert encoding novobiocin resistance (gyrase B; 2.4 kb).
Using In-Fusion Cloning, both scientists were able to efficiently clone their fragments into a vector linearized by restriction digest. Positive clones were determined through screening by colony PCR. While the specifics of each project had different challenges, the outcomes were the same: In-Fusion technology yielded positive clones on the first try with minimal hands-on time.
PCR amplification of the desired inserts provided single, distinct bands of the correct sizes (Figure 1). Subsequent cloning of these inserts into their respective destination vectors was analyzed via colony PCR (Figure 2). For Study 1, eight out of ten colonies resulting from the In-Fusion Cloning protocol were positive clones. For Study 2, 21 out of 32 screened colonies were positive clones. In both instances, researchers were able to show successful incorporation of their genes of interest.
“The kit exceeded expectations. PCR clean up kit yielded high DNA concentration (~90 ng/µl). Also, I have not been able to obtain any positive clones using the regular ligation method. Using [In-Fusion] cloning I was able to obtain 21 positive clones. Thanks for the help guys!”
Figure 1. PCR amplification of cloning inserts. The genes of interest were amplified from genomic DNA templates using CloneAmp HiFi PCR Premix. PCR primers introduced In-Fusion Cloning overlap sequences to the 5' and 3' ends of the product. (Panel A) Study 1, ~1-kb insert. (Panel B) Study 2, 2.4-kb insert.
Figure 2. Colony screening. Colony PCR was used to determine successful clones. (Panel A) In Study 1, eight out of ten colonies tested showed a band at ~1.3 kb, indicating a correct clone. (Panel B) In Study 2, 21 out of 32 colonies tested showed a band at ~2.6 kb, indicating a correct clone.
Whereas previous cloning efforts with ligation-based methods had failed to produce satisfactory results, In-Fusion HD Cloning Plus generated positive clones for two separate projects. In just one attempt each, both researchers were able to successfully clone their challenging inserts into pET expression vectors.
The gene of interest (~1 kb with 67% GC content) was PCR-amplified from 100 ng of genomic DNA template using the provided CloneAmp HiFi PCR Premix. Amplification primers were designed such that the 15-bp overlaps required for In-Fusion Cloning were added to both the 5' and 3' ends of the PCR product. Thermal cycling conditions were set per the recommendations in the user manual, with an extension time of 10 seconds, for a total of 35 cycles.
The destination vector was prepared for cloning via restriction digest. 2 µg of pET20b(+) was linearized with NdeI and XhoI for 1 hr at 37°C.
Both the PCR-amplified insert and linearized vector were purified using the included NuceloSpin Gel and PCR Clean-Up kit. As detailed in the In-Fusion HD Cloning Plus user manual, the PCR fragment was then cloned into linear pET20b(+) with the In-Fusion HD Enzyme Premix, and this mixture was used to transform the provided Stellar Competent Cells. Resulting colonies were screened by colony PCR to confirm successful clones.
The E. coli CC5 (NovR) (R136H) strain was obtained from the Mawell lab (Contreras and Maxwell, 1992) and grown overnight in LB without antibiotics. Genomic DNA (at a concentration of 3,561 ng/µl) was extracted from the overnight culture and used as PCR template for amplification of gyrase B using the CloneAmp HiFi PCR Premix provided with the In-Fusion HD Cloning Plus kit. Primers were designed to include the required 15-bp overlaps for In-Fusion Cloning reactions, and PCR cycling conditions were set according to the user manual, with an extension time of 150 sec, for a total of 30 cycles.
The PCR product was purified using the NucleoSpin Gel and PCR Clean-Up kit provided with the In-Fusion Cloning kit, yielding 95.1 ng/µl purified insert. The destination vector (pET28a) had been previously double digested overnight at 16°C with XhoI (Promega) and NdeI (Promega), with successful digestion confirmed via 1% agarose gel electrophoresis.
Using the In-Fusion HD Enzyme Premix, the purified insert was cloned into the linearized vector according to the protocol in the user manual. The cloning reaction was used to transform the provided Stellar Competent Cells, and cells were plated on LB + Kanamycin (30 µg/ml). Clones were analyzed via colony PCR using EconoTaq DNA Polymerase (Lucigen), and PCR products were run on a 1% agarose gel.
Contreras, A. and Maxwell, A. (1992) gyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase. Mol Microbiol. 6(12):1617–1624.