Rapid Cloning and Identification of Recombinant Plasmids Using the Fast-Link™ DNA Ligation and Screening Kit, Epicentre® (an Illumina® company)


	Contact Us: 800-284-8474 Log In

PRODUCTS RNA Sequencing Array-Based Analysis Nucleic Acid Purification & Extraction Kits End-Point PCR Reverse Transcription and cDNA Synthesis In Vitro Transcription and RNA Research Genomic Library Construction Kits, Gene & PCR Cloning Kits DNA and RNA Polymerases Phosphatases, Kinases, & Ligases Nucleases, Glycosylases, & DNA Binding Proteins Transposomics™ Glucosyltransferases Deadenylases WHAT'S NEW New Products TargetAmp™-Pico Labeling Kit for Illumina® Expression BeadChip® 5´ Deadenylase Pvu Rts1I Endonuclease T4 Beta-glucosyltransferase Ribo-Zero™ Magnetic Kit (Human/Mouse/Rat) ScriptSeq™ v2 RNA-Seq Library Preparation Kit Ribo-Zero™ Gold Kit (Human/Mouse/Rat) Ribo-Zero™ rRNA Removal Kit (Meta-Bacteria) Terminal deoxynucleotidyl Transferase, Recombinant EZ-Tn5™ Custom Transposome™ Construction Kits T4 RNA Ligase 2, Deletion Mutant TargetAmp™-Nano Labeling Kit for Illumina® Expression BeadChip® Meetings/Trade Shows News and Press Special Offers Scientific Posters TECHNICAL RESOURCES Protocols EpiCentral Technical Blog Request Help FAQ Newsletter Archives Request Literature DNA Sequences MSDS Technical Appendix ORDERING Log In/Register Ordering Info International Distributors Terms and Conditions Privacy Policy COMPANY Company Overview Contact Us International Distributors Location and Directions Careers Legal Information

Epicentre Forum 3 (2)

|Rapid Cloning and Identification of Recombinant Plasmids Using the Fast-Link™ DNA Ligation and Screening Kit|

Even with recent improvements in technology, DNA cloning is still a time consuming process. Increasing the speed and efficiency of this procedure can save both time and money. Using Epicentre's new Fast-Link™ DNA Ligation and Screening Kit, ligation of inserts containing cohesive overhangs or blunt ends can be performed in just five minutes. Ligation of PCR products with A-overhangs into T-vectors can be completed in one hour. Following ligation and transformation, recombinant screening using a gel lysis technique^1,2 allows screening of colonies for insert-containing plasmids in less than one day without having to grow cultures and perform mini-preps. In the following experiment we use the Fast-Link™ Kit to clone all three types of inserts and identify recombinants.

The following three ligation reactions were assembled, each in a total volume of 15 µl in 1X Ligation Buffer with 2 U of T4 DNA Ligase: 1) 100 ng of a 4.0 kb fragment cut with EcoR I and Hind III and 25 ng of pBSK+ cut with EcoR I and Hind III with 1 mM ATP; 2) 100 ng of a 1.5 kb Sma I fragment and 20 ng of EcoR V cut pBSK+ with 0.5mM ATP; 3) 150 ng of a 1.1 kb PCR product and 50 ng of pT7Blue T-vector (Novagen) with 0.5 mM ATP. The overhang and blunt ligation reactions were incubated at room temperature for 5 minutes. The T-vector ligation was incubated at 16°C for one hour. Each reaction was then incubated at 70°C for 15 minutes to inactivate the ligase. Following heat treatment, 2 µl of each reaction were transformed into 20 µl of E. coli NovaBlue cells (Novagen) according to the manufacturer's instructions. After a one hour outgrowth, the cells were plated on LB Amp/Tet/X-gal plates and the plates were incubated overnight at 37°C. The results are shown in Table 1.

Table 1. Ligation Efficiencies Obtained Using the Fast-Link� Kit.
	Ligation Time	% White Colonies	Recombinants per µg DNA
Overhang	5 min.	93	2.0 x 10⁶
Blunt	5 min.	71	4.4 x 10⁵
PCR product	1 hr.	68	1.2 x 10⁴

Twelve white transformants from each ligation were then screened for the presence of the insert using the gel lysis technique shown in Table 2.

Table 2. Fast-Link™ In-well Lysis Screening Protocol.

Pipet 15 µl of Protoplast Buffer into the well of a round-bottom 96 well plate.
Pick colonies with a sterile toothpick and swirl the toothpick in the Protoplast Buffer for 10-15 seconds.
Patch bacteria from the toothpick onto an agar plate containing the appropriate antibiotic(s). Use a grid so that each colony can be identified.
Incubate the agar plate at 37°C overnight. (See note, below.)
Incubate the 96 well plate at room temperature for 10 minutes to allow protoplast formation.
Add 4-8 µl of Lysis Buffer into the appropriate number of wells on a submerged agarose gel. The amount of Lysis Buffer required depends on the size of the well. The percentage of agarose used should separate plasmids containing inserts from plasmids without inserts. In a separate well on the gel, load a supercoiled kb ladder as a marker.
Add the entire contents of each plate well of proto-plasts to separate agarose gel wells containing Lysis Buffer and incubate for 15 minutes to allow the protoplasts to lyse.
Separate the DNA fragments by gel electrophoresis and stain the gel with ethidium bromide.

Note: After the plate from Step 4 incubates for 2-3 hours at 37°C, sufficient cells will be present from which small cultures of identified recombinants can be started. Alternatively, the screening toothpick from Step 2 may be used to start the cultures.

Screening of white colonies from the cohesive, blunt, and PCR ligation transformations yielded 100, 58 and 50% recombinants respectively (Figure 1). These percentages were used to calculate the total number of recombinants per microgram of DNA transformed. The results are shown in Table 1.

Figure 1. Results of recombinant screening using the gel lysis technique. Panel A - ligation of DNA containing cohesive overhangs; Panel B - blunt end ligation; and Panel C - ligation of PCR products.

We have shown that the Fast-Link & DNA Ligation and Screening Kit reduces the time necessary to clone and identify recombinant plasmids. The colony screening method is both faster and less expensive than standard screening methods such as PCR or plasmid preparation and subsequent restriction mapping.

1995 Epicentre Forum 2 (2), 6.
Sekar (1987) BioTechniques 5, 11.