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MOPS(cas 1132-61-2) for gene experiments

Reagents: TRIzol, chloroform, isopropanol, 75% ethanol (prepared with DEPC treated water), RNase water, coke carbonase diethyl ester (DEPC), MOPS(cas 1132-61-2), sodium acetate , Ethylenediamine tetraacetic acid disodium, formamide, 37% formaldehyde, glycerol, bromophenol blue, hydrochloric acid, sodium hydroxide, and agarose. Except for TRIzol purchased from GIBCO / BRL, DEP purchased from Sigma, the rest of the products are homemade analytical grade.

The preparation and analysis of RNA is essential for understanding gene expression and transcriptional regulation and cDNA synthesis. There are several ways to extract RNA. Combined with our research, introduced one of the simple, fast extraction method. The total RNA of the retina was obtained by extraction, which was used to further analyze the gene function of the gene from the transcription level of the gene, such as Northern Blot, RT-PCR and other molecular biology experiments.

Preparation of the organization: the rabbit retina tissue removed, set the sample bottle, -20 ℃ refrigerator. 

Experimental procedure: Tissue block homogenizer, add TRIzol (100mg tissue plus TRIzol 2mL), freeze homogenate, set Eppendorf tube. 15 ~ 30 ℃ for 5min, add chloroform (0.2mL chloroform / 1mL TRI zol), cover the lid, shake for 15s, 15 ~ 30 ℃ incubation 2 ~ 3min, 4 ℃ 12000r / min centrifugation 15min, take the supernatant to the new (0.5 mL isopropanol / mL TRIzol), 15 ~ 30 ℃ incubation sample 10min 4 ℃ 12000r / min centrifugation 10min, discard the supernatant, 75% ethanol washing precipitation once (at least 1mL 75% Ethanol or mL) at 4 ° C 7500 r / min for 5 min, discarded with ethanol, air or vacuum for 5 to 10 min (do not completely dry, do not vacuum centrifuge), add RNase (or 0.5% SDS) to dissolve RNA , with pipette suction several times. Preparation of 1% agarose denatured gel: 10 mL of 10 M MOPS buffer, 10 mL of 0.1% DEPC water, 1 g of agarose, heated to complete elution, cooled to 60 ° C at room temperature, 6 μl of ethidium bromide (10 mg /mL), 5.4mL 37K formaldehyde, mix, set the ventilation cabinet 15min, pour the plastic device after the glue. Prepare the sample 10 ~ 20μg, add 20μL loading buffer, 95 ℃ 2min denatured RNA, sample, 100V voltage electrophoresis electrophoresis, until the bromophenol blue to plastic downstream 3/4, observed in the UV transmission platform, Measuring 28S rRNA and 18SrRNA to the original hole distance, take pictures at the end.

 

 

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