experimental method
- Northern blot
- Northern Blot
| Principle of experimental method | The basic outline of Northern blot is to separate RNA samples by denaturing agarose gel electrophoresis, transfer them to a solid phase membrane carrier such as nylon membrane, and immobilize them on a solid phase membrane with radioactive or non-radiolabeled DNA or RNA-specific probe pairs. The mRNA is hybridized, the membrane is removed to remove the non-specific binding hybridization signal, and the hybridization signal is analyzed by autoradiography or color reaction. By comparing the migration position of the hybridized mRNA molecule with the standard molecular weight molecule in electrophoresis, the size of a specific gene transcript in the cell can be known; and the strength of the mRNA expressed by the gene can be known by comparing the strength of the hybridization signal. |
|---|---|
| Experimental Materials | DNA |
| Reagents, kits | MOPS electrophoresis buffer formaldehyde gel loading buffer EB EGFR probe DIG SSC |
| Instruments, consumables | Horizontal Electrophoresis System Ice Machine Constant Temperature Water Bath Gel Imaging System EP Tube Pipette Tip Head |
| Experimental procedure | First, RNA transfer and fixation 1. After the total RNA sample was separated by denaturing agarose electrophoresis, the gel was rinsed with 1×MOPS buffer for a while, and the equilibrium gel was soaked in 10×SSC for 30 min. 2. Transfer the total RNA to the nylon membrane by DNA transfer method and apparatus in the southern blot experiment (with RNase-free operation in the process). 3. After washing the nylon membrane with 1×SSC, blot the filter paper and bake it in a 2-layer clean filter paper for 8 hours in an oven at 80 °C. 4. RNA staining, dried nylon membrane was immersed in 5% glacial acetic acid for 15 min at room temperature. 5. Soak for 5 to 10 minutes in 0.5 mol/L sodium acetate and 0.04% methylene blue solution. 6. Deionized water is rinsed for 5~10 min. RNA standard and bands of 28s and 18 sRNA are visible, and the soft pencil is marked. Second, pre-hybridization, hybridization and color development 1. Washing buffer 1 time (3~5 min). 2. 100 ml blocking buffer working solution is blocked for 30 min. 3. Incubate for 30 min in 100 ml antibody buffer (1:5000). 4. Washing buffer wash film (15 min × 2). 5. 20 ml detection buffer Balance for 2~5 min. 6. Place the nylon membrane in a 10 ml color-substrate solution brown bottle, protected from light for a few minutes to one day (do not shake when color appears). 7. When a strip appears, the TE-buffer washes the membrane (5 min × 1) and terminates the color. 8. Photographic recording, dried at 80 ° C, stored. 9. Scan to calculate the fold of the EGFR gene amplification. |
| Precautions | 1. Avoid RNase contamination and prevent RNA degradation. 2. It is best not to add EB to the gel to avoid reducing the efficiency of hybridization. 3. In order to reduce the membrane hybrid background, the pre-hybridization time can be appropriately extended. |
Sweet corn. The light green outer leaves of sweet corn are yellow grains, but also purple and yellow. The grains are small and round, the skin is thin and soft, tender, sweet and delicious.Contains carotene, zeaxanthin, with the effect of eye protection.
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