Rt-pcr reversetransThe criptace chain reaction is designed to design dna polymerase, intellectual background: 1 - genetic expression: dna rna protein has a gradual amplification mechanism for the expression of single-copy genes, such as 104 silky meringues of proteins (4d per mrna survives with 105 silk proteins). So the mrna expression level of a single copy gene is important for the regulation of its functional level. Pcr technology (olymerase chain reaction). Under the conditions of the presence of templates, quotations and four deoxynucleic acids, reliance is placed on the enzyme reaction of dna polymerase, the specificity of which is determined by two artificially synthesized quotation sequences. Reaction in three steps: a
2. Breaking the hydrogen key of the double-heated dna to form a single chain of dna by heating c. Extension: with the presence of dna polymerases and dntps and mg2+, deflammants extend in the direction of 5 3. The above three steps are a cycle, so repeated. 3. Reverse transCriptase) is a dna-dependent polymerase that exists in the rna virus and has at least three types of activity: the rna-dependent dna-polycing activity: the rna template is used to synthesize the cdna first chain; the rnace hydrolytic activity: the rna 3 in the hydrolysis rna hybrid, the dna-dependent dna-polysing activity: the preparation of the double-chain cdna. Ii, rt-pcr, which is complemented by the first dna chain:
3, 1, the design of the quotation and its principles: 1) the specificity of the quotation determines the speciality of the reaction of the pcr. Therefore, the rationality of the design of the quotation has a crucial impact on the entire experiment. In the design of the quotations, due consideration is given to the possible congener sequences, the different subtypes of the same protein, the different mrnai formulas and the possible hnrna effect on the specificity of the quotations. Select as much as possible to overwhelm the quotations of the two inlines, or the inlines that exist during the target protein expression but do not exist in other subtypes. 2) management of citation design principles: the citation design principles include a, length of citation: in general, 1530 bp, too short the quotation will affect the speciality of the pcr, and too long the maximum extend temperature of the pcr will exceed the maximum temperature of the taq enzyme and affect the speciality of the reaction. B. Base distribution: the four bases should preferably be distributed at random to avoid a concentration of groaning or secreting
4 in particular, there are more than three consecutive single base bases. Gc content (tm): 40% 60% and pcr amplified compound temperature is typically lower tm minus 510 degrees. C. 3-end requirement: 3 the end must be strictly complementary to the template, not subject to any modifications or the possibility of creating any secondary structure. The last alkali base is the least triggerable of error at the time of a, and the g c is in the middle, so the three ends of the quote are best placed to push g-c and avoid, as far as possible, the occurrence of more than two tods in succession, with its own secondary structure: it should not have a series of complementarities, otherwise it would fold itself into a hairline structure or its own compound. E. Secondary structure between quotations: not more than four successive base-based complementarities between quotations, 3, end not more than two. F. Congener sequences: the homogeneity of quotes and non-specific augmentation sequences should be less than the complementary base presence of eight consecutive entries. G. 5-point unrestricted: 5
5 the end base base can be separated, but it would be preferable for g or c to stabilize the end of the pcr product. They can also perform special decorations (marks, enzymes, temples). Select appropriate quotations for experimental purposes. Common quote design software, e. G., equals these conditions can be set on your own. 2 consumption material: the materials used in experimental exposure samples, such as cold storage tubes, guns and ep tubes, are pre-empted by %de p-water leaching, removing rna enzymes and preventing degradation of rna during operation. It is then subjected to high-pressure eradication (sterilisation and active decpo3, reagent preparation: degenerative fluids, water saturated phenol, sodium aceticate, chloroform, isopropanol, 75% oscillation, treated by decp and high pressure water. , rna spoon extraction method: the quality of rna separated from cells in rt-pcr is critical, including the purity and integrity of the rna. The most critical factor in rna separation is to minimize contamination of rna enzymes. But rn..
6 and a enzyme activity is very stable and widely distributed, with a large number of rna enzymes in the environment in addition to the intracellular rna enzymes. Therefore, in the extraction of rna inches, an environment free of rna enzymes should be created to the greatest extent possible, including de-source rna enzymes contamination and inhibiting endogenous rna enzymes activity, mainly by quenching corocarbonate diethylene (depc de-sourced rna enzymes), inhibiting endogenous rna enzymes by inhibiting protein rnasin and powerful protein variants such as iso-sulphates. Experimental operating procedures, experimental devices and materials: 1, liquid guns: 1 ml, 200 卩 1, 20 卩 1, 10 卩 1, 2 卩 12, suction head: 1 ml, 200 卩 i, 20 卩 13, slurry pipe: 5 ml4, snort table: one of the meml inhaled head, one of the five, ep pipe: 100 卩 16, reagent bottle: 2 60 m
7-l brown reagent bottle (blank, cover) 1-125 ml white reagent bottle (free ethanol) 7-l: 250 ml, 250 ml, 500 ml & capacity bottle: 250 ml 500 ml 1000 ml, test stand: 5 ml., 20,110, salt water bottle: 250 ml. 500 ml each, one waterless ethanol, one water-free lunch box: 4 12 aluminium lunch box: 13, large porcelain tank: 2 14, tin plum paper: 15, 2 rolls of paper, 16 triangles: cap, slightly larger, treatment of experimental equipment and preparation of 1 plastic (including gunhead, ep tube, plaster tube, etc.) first pours the water from the capacity bottle into a porcelain tank, with plastic immersed in it: 15, rolls of paper: 2 rolls of paper, and then drying it, then drying it, drying it with the first hand of a gun, then draining it to the front of the capacity bottle
8 , ep2, glass products: smelting acid over the night, washing clean, drying-up (depc bubble) (cleaning 1%). Depc spends the night and then drys it)3 and the slurry: (including scissors, shrubs) wash first and then press high pressure (no bubbles in depc, reagent formulation: stand by for four hours in 1000 ml capacity bottles. 1 depc water: 1 ml in 1000 ml double steam water for 1% depc water, 2 ethanol, 75% ethanol: with water-free ethanol depc water, then 20 c (i= epc water with high pressure), 3 isopropanol: 4 in brown bottles, chloroform: 5 in brown bottles, 4 in glucose, formulation of several buffer fluids, 1 in electro-brain: tris 54 gperate edta 20ml? Vapour water 1000 ml5xte (stored fluid)
9. Multi-accomplishments can be used in electron swimming (i. E. Concentration of work fluids), such as the 50ml storage liquid 450 ml water for 1,500 ml z as buffer 2 and the upper buffer: %dibenzophene ff 30% glycerine 6x buffer fluid, 4c preservation of 5th, gel gel formulation 1 and % glucose 100 ml electric swimming buffer fluids, 30 seconds to boiling in microwave furnaces, melted resin cooling up to 60 c with 10 mg/ml desertized acetene at 60 c, 卩1, fully equilibrified, pouring thermogels into well-laid membrane, now swimming after 30-45min placement in room temperature. 2, %: idem, change the volume of gum to 6, rt-pcr materials to be purchased: (breeders. Tel. 2236106.) taq enzyme (including mgci2 buffer) 200udnt p: 1 oligo (dt) 15:10d
10, promegam-mlv: 1 promega (buffer) rnasin: 1 11020 codepc 5gtrizol 100ml/l bottle invitrogen life technologies 4cmarter: 10 pg g/ml kovai (1543. 994. 697. 517. 371. 237. Vii, initiation justice 5 - cacgattgaggaggattcatc - counter-effect: 5 - taaagactctctcacacacact-2, p4: justice: 5 - gggactcgacgatracacacacac - counter-effective: 5 - tgttcctcggaggaggag-3
11 calculation of 2 (at) 4 (gc) positive and negative averages, with upward and downward fluctuations (ground 4c, or earth 504, each of which is combined with 5od, each of which is divided into 5d, gravity dilution: n depc water volume of 2? Nmol / 0d x numeric number of 0d on the tube x is 8 in 10p mol / 卩1 concentration, pcr product electron swimming, first taking about 1-10 卩1 pcr products, plus aluminum acid placed on paper, repeated insulation and subsequent electron swimming, general current of 10 ma 100 v, 30 minutes of electric swimming, observation under ultraviolet lamps, satisfactory results scanning printing. Ix. Keys one: the key step for reversal is immediate ice baths? At 2, rt, open pcr preheat for 30 minutes. Fellw delete i always hold a centrifuge cold. Cell ix 107 100 mg plus mlt
12, rizol cells blow up liquids with an inil emitter to clarify and transfer to the ep tube (repeated and then transferred to the ep tube) inverted flats of 10 under 10 (organizing 100 mg/ml per ep tube), room temperature reverses 10 mm/mic1/5 volume (must be in total volume) 5 minutes 4c, centrifugal 12,000g, 15 minutes up to layer (about 400 卩i) in another tube (about 400 卩i), 10 minutes air drying up to 20 卩i1-20i (10 卩i) in pc water, and 75% of ethanol cooling with ice (one ml4c) in 10 minutes (can be removed from the heart 7500g), 5 minutes clean air drying to 20 卩i (10 卩i-20i) in pc water (not fully dried)
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