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Pre-hybridization-free Northern blot hybridization solution and application thereof

A hybridization solution and pre-hybridization technology, which is applied in the field of molecular biology, can solve the problems of increasing the length of the experiment and consuming the energy of the experimenter, and achieve the effect of saving experimental time, reducing experimental steps, and promoting the combination of probes and RNA

Active Publication Date: 2022-05-31
SHENZHEN UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the Northern blot experiment, it is an essential experimental step to hybridize the probe to the membrane with RNA. The hybridization solution and the membrane are pre-hybridized, and the pre-hybridization step takes 0.5-4 hours, which seriously increases the length of the experiment and consumes the energy of the experimenter

Method used

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  • Pre-hybridization-free Northern blot hybridization solution and application thereof
  • Pre-hybridization-free Northern blot hybridization solution and application thereof
  • Pre-hybridization-free Northern blot hybridization solution and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] The hybridization solution was prepared according to the optimal formula of the Northern blot hybridization solution in Table 1.

[0024] Table 1 Optimal formulation of Northern blot hybridization solution (pH7.4, 1L)

[0025]

[0026] Reagent preparation instructions:

[0027] 1.20×SSC: mix 175.3g NaCl and 88.2g sodium citrate 2H 2 Dissolve O in 800 mL of deionized water, adjust the pH value to 7.4, add deionized water to make up to 1 L, sterilize at high temperature and high pressure, and store at room temperature.

[0028] 2.Northern blot hybridization solution: Dissolve Na with 200mL sterile water 2 HPO 4 , after adjusting the pH value to 7.4 with phosphate, then add other corresponding reagents to dissolve fully, and then dilute to 1L.

[0029] 3. After the Northern blot hybridization solution is prepared, the precipitate can be dissolved in a warm bath at 65°C before use, and it can be stored at room temperature or 65°C for a long time.

Embodiment 2

[0031] U6 was used as a probe, and the hybridization solution of Example 1 was used for experimental verification. Extract total RNA from Arabidopsis thaliana by Trizol method, denature the sample RNA, add denaturing gel for electrophoresis, and then perform membrane transfer and cross-linking, perform different hybridization treatments (see Table 2 for experimental design), membrane washing, and biotin labeling , wash the membrane, and finally detect by autoradiography.

[0032] Different hybridization treatment design: In this experiment, 5 groups were designed as 1#, 2#, 3#, 4#, 5#, and each group was loaded with 10ug, 5ug, 1ug ​​of total denatured RNA.

[0033] The hybridization temperature was 50°C for different hybridization treatments.

[0034] For different hybridization treatments, the volume ratio of U6 probe (0.25 μM) to hybridization solution was 1 ul: 1 mL.

[0035] Table 2 Example 2 Experimental scheme design

[0036]

[0037] The results show that the hybr...

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Abstract

The invention discloses a pre-hybridization-free Northernblot hybridization solution and application thereof, the pH value of the pre-hybridization-free Northernblot hybridization solution is 7.4, and the pre-hybridization-free Northernblot hybridization solution is prepared from the following components with final concentration: 5 * SSC, 20mM Na2HPO4, SDS (sodium dodecyl sulfate) with the mass percent of 7%, polyvinylpyrrolidone with the mass percent of 0.02%, polysucrose 400 with the mass percent of 0.02%, BSA (bovine serum albumin) with the mass percent of 0.02% and Tween-20 with the mass percent of 0.5%. And the 5 * SSC contains 0.75 M of NaCl and 0.075 M of sodium citrate. By adopting the Northernblot hybridization solution disclosed by the invention, the probe can be directly added into the hybridization solution without a pre-hybridization step of the hybridization solution and a membrane, so that the probe is hybridized with RNA (Ribonucleic Acid) on the nylon membrane, and the experiment steps and time are reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a pre-hybridization-free Northern blot hybridization liquid and application thereof. Background technique [0002] Northern blot is one of the important means mainly used to detect changes in gene mRNA expression levels. Compared with other detection methods of gene expression (such as QPCR, gene chip, RNase protection experiment, etc.), Northern blot has the characteristics of high sensitivity, strong specificity and low false positive, which makes Northern blot experiment considered the best method for detecting relative expression of genes. Authentic and reliable experimental methods. During the Northern blot experiment, it is an essential experimental step to hybridize the probe to the membrane with RNA. The hybridization solution is pre-hybridized with the membrane, and the pre-hybridization step takes 0.5-4 hours, which seriously increases the length of the ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6813
CPCC12Q1/6813C12Q2527/125
Inventor 白永生徐驰莫蓓莘唐玉林
Owner SHENZHEN UNIV
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