Describe technology of non-surgical embryo extraction on cattle
Non-surgical extraction of embryos is performed by the following way. A flexible catheter with an inflatable cuff is inserted into the vagina and through the cervix in one of the horns of the uterus. The cuff is inflated and closes the caudal exit of the horn of the uterus, thereby restricting a washing cavity. The catheter can be two-channel, which allows flow through the washing liquid. When using a single-channel catheter, the wash fluid is injected several times (5-8 times) and then flows out of the horn of the uterus. In both cases 200-300 ml of Dulbecco's phosphate buffer are introduced. Most optimal time for the extraction of embryos - 6-8 day after the start hunting, since early blastocysts of this age are most suitable for deep freezing and can be highly effective transplanted non-surgically. The donor cow is 6-8 once a year, extracting 3-6 embryos.
Materials which are used to determine the nucleotide sequence of DNA witch CEQ 800 analyzer
Standard set of reagents for cyclic sequencing using CEQ dye-terminators WellRED partially mixed (CEQ DTCS - Quick Start Kit); reaction buffer, dNTP mix, dye-labeled terminators, DNA polymerase are supplied in one test tube as the main mixtures; control matrix PUC18; Highly purified DNA sample pUC18 (a double-stranded plasmid with a length of 2,685 base pairs); primer for sequencing-47; glycogen; coating solution sample (Sample Loading Solution, SLS); mineral oil; deionized and sterilized water; 95% and 70% ethanol solution in water; 3M sodium acetate solution, pH 5.2; 100 mM Na2EDTA solution, pH 8.0; the second component of the stop solution; sterile microcentrifuge 0.5 ml tubes; thermocyclable thin-walled test tubes a volume of 0.2 ml or a microplate; thermal cycler with heated cover.
The principle of determining the nucleotide sequence of DNA in CEQ 800 genetic analysis system
In the CEQ ™ genetic analysis system 8000 for the determination of the nucleotide sequence of DNA the technology of capillary electrophoresis is used. Fragments of DNA, having a net negative charge, under the action of The applied fields are moved to the anode (+). At the same time, separation of DNA fragments, since migration is accompanied by sieve effect on the size of migrating molecules. Detection dye-labeled fragments are carried out at the signal fluorescence, then the signal is processed and fragments are represented as a nucleotide sequence. As solution of the electrolyte, which is a separation medium, polyacrylamide gels are widely used. The Genetic Analysis System CEQ ™ 8000 is self-contained complex designed to determine the nucleotide sequences and sizes of DNA fragments in samples, prepared for analysis with the help of Beckman Coulter reagents, containing a fluorescent label-dye. For determining nucleotide sequences using dye- terminators a set of four-color reagents is used, for analysis of the size of fragments - primers labeled with a dye. The system consists of three main parts: chemical, hardware and software. In one plate, up to 96 samples can be placed, each of which is labeled with four dyes. Eight samples (set samples) in each strip of the plate containing labeled fragments DNA, are automatically denatured when applied. Then the separation by means of capillary electrophoresis. Gel is automatically replaced after each separation. For filling capillary gel in the device is installed a special cartridge, volume of which allows one to analyze one 96-well the tablet. The detection of signals induced by lasers occurs according to four spectral channels. Primary data for each The capillaries are automatically processed. After analysis is complete the user is provided with either a sequence of nucleotides in text form, or a list of fragments.
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List individual components of the CEQ 800 analyzer.
Various components of the device are responsible for the process of preparation of samples, the performance of separation and the phase of detection of the signal.
The sample separation cover is transparent. In this compartment there is a plate for the buffer, a container for wetting the capillaries and a plate for the samples.
The lid of the capillary compartment provides access to the capillaries. This compartment can be closed with a thermostatic cover.
The thermostating cap of capillary separation allows to create the necessary temperature in the capillary compartment.
Block of capillaries. Capillaries have a length of 33 cm with an internal diameter of 75 μm. For operation with capillaries, the CEQ 8000 analyzer includes a unique plenum.
The capillary block consists of:
an electrode block, eight capillaries and a fitting.
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Tablet holders are used to fix the plate with digestible samples, a plate for the DNA separation buffer with a lid that prevents evaporation, and a container for moistening the capillaries. In the capillary moisturizing container containing deionized water, the capillary ends are immersed. In a bottle for the used gel, a gel is collected from the collector when cleaning the system.
The gel pump is used to fill the capillaries with fresh gel from the cartridge, sufficient to analyze one set of samples or a 96-well plate.
The gel cartridge contains a fresh gel for the separation of DNA. The filled cartridge contains a sufficient amount of gel to perform 96 separations (12 fillings of the capillary block). If the gel cartridge is not installed, for example, when transporting or storing the device, a plug is inserted in the pump casing to prevent the gel from drying out.
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