Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

The overall goal of the following experiment is to construct gene targeting vectors using sub cloning plus insertion. This is achieved by transforming the back containing e coli with a recombining plasmid. The second step involves generating asymmetric phosphorolated insertion cassettes, and sub cloning plasmids by polymerase chain reaction.

Following the addition of arabinose, the GBAA recombining proteins are expressed and the terminal modified insertion cassette and sub cloning plasmid are electroporated. The desired DNA sequence is simultaneously captive from the back and modified with the insertion cassette into the sub cloning plasmid. The main advantage of this technique of existing methods like standard recombining, is that gene targeting vector construction can be performed in a single reaction.

To begin design oligos for the insertion cassette and the sub cloning plasmid design each oligo such that it contains 180 base pair homology arms flanking the genomic target site and 20 base pairs a specific priming sequence for the insertion cassette or sub cloning plasmid. Next, determine the orientation of the genomic DNA insert of the back clone using a web-based tool like clone db. Establish the direction of replication from or ES by checking the map of the back plasmid backbone used in the back.

Library construction, add two terminal phospho eight bonds, so the five prime end of the oligo that is opposite the direction of replication on the back clone. Next, add a five prime phosphate modification to the reverse oligo. First stab a sterile pipette tip into the back agar culture and inoculate 5.0 milliliters of odine broth.

Grow the culture at 37 degrees Celsius for five hours with shaking at 200 RPM. Next, chill, 10%glycerol solution, micro centrifuge, tubes and electroporation, cuvettes on ice cooler, refrigerated large centrifuge and micro centrifuge to four degrees celsius. After incubation, determine the optical density of the culture using a spectrophotometer.

Measuring absorbance. At 600 nanometers. Prepare the electro competent cells when an OD 600 reading of 0.3 to 0.8 is reached.

When the culture is ready, spin down the cells in a 50 milliliter centrifuge tube. Wash the cells with one milliliter of chilled 10%glycerol and spin again. Perform these wash steps a total of three times.

Resuspend the cells in a total volume of 50 microliters and add 10 to 200 nanograms of the recombining plasmid. Obtain a single cell suspension by pipetting up and down several times, and then transfer the cells to a pre chill electroporation.Vete. Next, place the vete into an electroporation device and electro operate the cells immediately recover the cells in one milliliter of LB and transfer the cells to a 50 milliliter centrifuge tube.

Grow the back culture at 30 degrees Celsius for two hours with shaking at 200 RPM. After this time, add nine milliliters of LB to the recovered culture. Grow overnight at 30 degrees Celsius with shaking at 200 RPM to incorporate the homology arms into the insertion cassette and the subc cloning plasmid.

Perform PCR using the long modified oligos prepared earlier. Alternatively, linearize the plasmid template Using a restriction enzyme, choose one that cuts outside the PCR amplification region and is heat in activated. Set up the PCR using a high fidelity hot start, DNA polymerase system.

Prepare a PCR master mix and perform thermal cycling according to best practices. After the reaction, analyze PCR products by AROS gel electrophoresis. Load one to five microliters of each PCR onto a 1%aros gel.

Next, purify the PCR products using a PCR purification kit. Quantify the PCR amplified DNA against a known set of DNA standards or by using a NanoDrop spectrophotometer. After an overnight incubation, dilute the back GBAA culture 50 fold and prepare a separate culture for a negative control.

Grow the diluted cultures at 30 degrees Celsius with shaking at 200 RPM for one hour and 50 minutes. Next, chill all recombining materials and equipment to four degrees Celsius as demonstrated earlier. Prepare a 10%weight per volume solution of OSE and filter sterilize through a 0.2 micron syringe filter.

When the OD of the back culture reaches the desired level, proceed with inducing the recombining proteins. Add 200 microliters of the 10%Arab solution to 10 milliliters of the back culture to achieve a final aose concentration of 0.2%Include an un induced culture to be used as a negative control. Transfer the cultures to a 37 degrees Celsius shaking incubator and induce red expression for 45 minutes with shaking at 230 RPM.

Spin down the cells and wash with 10%glycerol three times as shown earlier at 600 to 1000 nanograms. Each of the insertion cassette and the sub cloning plasmid to the recombining reaction include a vector only and vector plus single insert control to check the recombination proficiency and integrity of the vector. Next, obtain a single cell suspension and electro operate the cells as shown earlier.

Allow recovery in LB media at 37 degrees Celsius for one hour for multi copy plasmids or in 10 lb at 37 degrees Celsius for three hours for back vectors. Complete different dilutions with the recovered culture on dual selection. Agar please and grow at 37 degrees Celsius for 16 hours.

When ready, pick colonies into five milliliters of LB containing the selective antibiotic and grow overnight at 37 degrees Celsius. Next, use a column purification kit to prepare mini prep DNA, introduce the appropriate restriction enzyme and then identify the clones containing the expected fragment sizes by gel electrophoresis. Finally, perform DNA sequencing across the homology arms and insertion cassette to check for oli photosynthesis errors.

In this example, restriction digestion analysis of P two RX one EYFP insert containing clones showed the expected pattern indicating that they contain the correctly assembled knockin vector. This schematic illustrates the concept of bact trimming. Using sub cloning plus insertion analysis of PCR products confirmed that cassettes were correctly incorporated.

In addition, when positive back clones were analyzed after adding the appropriate restriction enzyme, the expected pattern was obtained. Restriction digestion analysis of 12 said R SR two SBI clones showed correct vector assembly in the majority of the recombinants. After watching this video, you should have a good understanding of how to construct gene targeting vectors rapidly using sub cloning plus insertion.