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The overall goal of this procedure is to make genome wide single gene deletions in streptococcus sanguine by high throughput PCR. This is accomplished by first obtaining three amplicons by high throughput PCR amplification, including the antibiotic resistance gene, as well as the upstream and the downstream DNA sequences of the target gene. The second step is to obtain the linear recombinant PCR amplicon using three PCR amplicons as templates.

Next, the competent s sanguine cells are prepared. The final step is to transform the linear recombinant PCR amplicon into competent s sanguineous cells. Ultimately colon A PCR amplification and DNA sequencing are used to verify the correct mutants.

The main advantage of this technique over exhausted other method like suicide plasmid knockout, is this method can create the genome wide deletion mutant directly use high throughput. PCR Primers can be designed and synthesized as described in the written protocol using in-house scripts based on the streptococcus sanguineous SK 36 genome sequence. Three sets of primers were designed for amplification of the one kilobase upstream sequence of the target gene, the A PHA three gene ENC coding, kanamycin resistance protein, and the one kilobase downstream sequence of the target gene.

Gene specific primers. R one and F three are designed based upon the five prime and three prime sequences of the target gene to high throughput amplify the one kilobase upstream or downstream region of the target s sanguine genes. First assemble a PCR cocktail mixture on ice in a 15 milliliter conical tube.

See the text for the components of the PCR mixture containing s sanguineous genomic DNA transfer 23 microliters of the mixture into each well of a 96 well PCR plate using a multi-channel pipette for the upstream sequence of the target gene transfer one microliter each of F1 and R one from 10 micromolar working primer plates to the PCR plate using the multi-channel pipette. Do the same with F three and R three primers to amplify the downstream sequence of the target gene. Seal the PCR plate and perform amplification at 94 degrees Celsius for one minute, followed by 30 cycles of 94 degrees Celsius for 30 seconds.

54 degrees Celsius for 30 seconds and 68 degrees Celsius for 1.5 minutes. Following amplification, prepare a 1%aros gel containing a fitting bromide with 48 wells fitting multichannel pipette loading. Mix four microliters of the PCR product with one microliter of five XDNA loading buffer.

On a 96 well plate load the samples on an aros gel using a 10 microliter multi-channel pipettes. Run electrophoresis at 135 volts for 30 minutes. Examine the band on gel under a UVP documentation and analysis system.

Purify the PCR products by the pure link 96 PCR purification kit using centrifugation according to the manufacturer's instructions for eluding DNA. Add 40 microliters of sterile double distilled water to each well of the binding plate. Randomly pick several purified amplicons on the plate to examine the DNA concentrations.

Using a NanoDrop spectrophotometer, adjust the concentration of amplicons on the plate to approximately 10 nanograms per microliter. Next, use eco R one to digest a plasmid containing the kanamycin resistance gisette so that it can serve as the PCR template. Purify the digested plasmid using the kayak quick PCR purification kit and adjust the linear plasmid DNA to final DNA concentration of 10 nanograms per microliter.

Assemble a 25 microliter PCR mixture for the kanamycin resistance Cassette amplicon, including F two and R two primers as listed in the written procedure. Accompanying this video, proceed to perform PCR amplification as instructed here. To examine the PCR product, perform gel electrophoresis on a 1%aros gel.

Then pool a bulk of the cany resistance cassette amplicons from 10 PCR purified individuals. Adjust the amplicon concentration to 10 nanograms per microliter. The single most difficult aspect of this procedure is obtaining the final linearly competent PCR applicant from its three PCR applicant t.

To ensure success, the three PCR applicant ter are adjusted to near equal amount. Combine one microliter each of the three 10 nanogram per microliter PCR aons in one PCR plate, as well as the PCR template. Then prepare the PCR mixture, including F1 and R three primers as listed in the written procedure.

Next, perform PCR amplification of the samples. Examine the PCR AMPLICONS on an aros gel following purification and quantification of the PCR amplicons. As before, freeze the recombined PCR Amplicons at negative 20 degrees Celsius.

To begin, prepare Todd Hewitt and horse serum medium as instructed in the written procedure accompanying this video aliquot two milliliters of medium and 10 milliliters of medium into two 15 milliliter conical tubes. Inoculate five microliters of thawed stock s sanguine SK 36 that had been frozen at negative 80 degrees Celsius into two milliliters of medium. Incubate the tightly capped culture overnight at 37 degrees Celsius.

Also pre incubate the 10 milliliter medium tube under the same conditions after overnight incubation. Transfer 50 microliters of the culture into the 10 milliliter medium tube. Incubate the tube at 37 degrees Celsius for three hours.

Immediately following incubation, add two microliters of 70 nanogram s sanguineous, SK 36 competence stimulating peptide and two microliters of linear recombinant PCR amplicon to einor tubes on a 96 well block and prewar them at 37 degrees Celsius. Transfer 330 microliters of the three hour incubated SK 36 culture into each tube. Replace DNA with sterile double distilled water as a control.

Incubate the tubes at 37 degrees Celsius for one hour following incubation. Place the block on ice spread 100 microliters of each transformation on brain heart infusion or BHI agar plates with 500 micrograms per milliliter of can mycin incubate the plates at 37 degrees Celsius for two days under micro aerobic conditions for each replacement mutant, randomly pick up two individual colonies and inoculate each colony into five milliliters of BHI containing 500 micrograms per milliliter of cany. Incubate each inoculum overnight at 37 degrees Celsius under micro anaerobic conditions.

Cryopreserve each culture in 30%glycerol at minus 80 degrees Celsius. To examine whether the mutant contains the expected gene replacement. Perform colony PCR for each mutant using F1 and R three primers in a 96 well PCR plate.

Use about one microliter of overnight culture from each individual colony as the DNA template in a 25 microliter PCR amplification reaction. Proceed to perform PCR amplification to confirm mutant gene replacement to precisely identify double band mutants or contaminant PCR amplicon. Examine the PCR amplicon by gel electrophoresis for four hours on a greater than 12 centimeter long.

2%aros gel with a th bromide staining under this agros gel electrophoresis condition. Any amplicons with greater than or equal to 100 base pair difference can be clearly identified. Alternatively, when bands resulting from amplification of the kanamycin resistance cassette and the wild type gene are anticipated to differ by less than 100 base pairs, use an internal T one primer of the target gene to determine whether a wild type gene can be detected by PCR.

To further confirm the deletion, purify the amplicons by pure link 96 PCR purification kit and sequence using the P one primer, which binds to the kanamycin resistance cassette, keep only the correct mutants confirmed by sequencing after PCR amplification using primers F1 and R one for the upstream sequence and F three and R three for the downstream sequence. Approximately one kilobase upstream and downstream of each s sanguineous gene were obtained in 96. Well format under the PCR conditions and using the designed primers.

A specific product was amplified from s sanguineous genomic DNA in each PCR reaction, which indicated the primers were highly specific to the targets of s sanguine through PCR re amplification using primers F1 and R three and three amplicons as DNA templates. Linear recombinant PCR constructs were created with high throughput. On a 96 well plate composed of a region upstream of each target gene, a cany cassette, and a region downstream of each target gene in that order, the recombinant PCR constructs were then transformed into s sanguine SK 36 and selected by cany on BHI agar plates.

For the majority of transformations, over 1000 colonies were obtained on each plate following a micro aerobic two day incubation. When these colonies were PCR amplified using F1 and R three, a single DNA band corresponding to the expected mutant size was observed by gel electrophoresis when attempting to delete potential essential genes. No colonies should be obtained in the majority of transformations for deletion of some essential genes.

However, colonies were still obtained following transformation after PCR amplification of these colonies using F1 and R three 2D NA bands corresponding to the expected mutant size and the wild type size appeared as revealed by gel electrophoresis. For some mutants, the expected mutant size and the wild type size of PCR amplicon were too close to be separated by long agros gel. In this case, an internal T one primer of the target gene was designed based upon the wild type gene sequence.

PCR was performed using the internal T one primer and F1 primer. The wild type SK 36 was used as a positive control strain for this PCR. If a band with the expected size was amplified from the mutant using the internal primer, the gene was identified as an essential gene.

Since the presence of can mycin cassette in the mutant was confirmed previously by DNA sequencing using the P one primer. Based on this protocol, 2048 non-essential S sanguineous gene mutants were collected. Excluding those four open reading frames contained entirely within other open reading frames.

218 genes, which are essential for s sanguine survival were identified under the experimental conditions. After watching these videos, I think you should have a good understanding how to make the genome wide. Gene ions use high throughput PCR.