Molecular cloning chapter 07Word格式.docx

1、This protocol, a variation of the method described in Chapter 7, Protocol 1 , involves lysis of cells in a monophasic solution of guanidine isothiocyanate and phenol. Addition of chloroform generates a second （organic） phase into which DNA and proteins are extracted, leaving RNA in the aqueous super

2、natant. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 g/mg starting tissue or 5-10 Protocol 3: Selection of Poly（A）+ RNA by Oligo（dT）-Cellulose Chromatography Chromatography on oligo（dT） columns is the preferred method for large-scale purificati

3、on （25 g） of poly（A）+ RNA extracted from mammalian cells. Typically, between 1% and 10% of the RNA applied to the oligo（dT） column is recovered as poly（A）+ RNA. Because the method can be frustratingly slow, it is not recommended for purification of poly（A）+ RNA from multiple samples. For this purpos

4、e, batch elution （ Chapter 7, Protocol 4） is the better choice. IMPORTANT:Protocol 4: Selection of Poly（A）+ RNA by Batch Chromatography When many RNA samples are to be processed or when working with small amounts （50 g） of total mammalian RNA, the technique of choice is batch chromatography on oligo

5、（dT）-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT:Protocol 5: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels Separation of RNAs acc

6、ording to size is the first stage in northern blotting and hybridization. The method described in this protocol uses glyoxal to denature the RNA, ethidium bromide to stain it, and agarose gel electrophoresis to separate the resulting glyoxal-RNA-ethidium adducts. IMPORTANT:Protocol 6: Electrophoresi

7、s of RNA through Agarose Gels Containing Formaldehyde Separation of RNAs according to size is the first stage in northern blotting and hybridization. The method described in this protocol uses formaldehyde to denature the RNA, ethidium bromide to stain it, and electrophoresis through agarose gels co

8、ntaining 2.2 M formamide to separate the resulting formaldehyde-RNA-ethidium adducts. IMPORTANT:Protocol 7: Transfer and Fixation of Denatured RNA to Membranes This protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow

9、of neutral or alkaline buffers. RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers. However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. IMPORTANT:Protocol 8: Northern Hybridization This protocol describes ho

10、w to carry out northern hybridization at high stringency in phosphate-SDS-buffers. Although a wide variety of formats are available, hybridization is usually performed in heat-sealable bags, roller bottles, or plastic boxes, as described here.IMPORTANT:Protocol 9: Dot and Slot Hybridization of Purif

11、ied RNA Dot blotting of RNA is best carried out using purified preparations of RNA that are denatured with glyoxal or formaldehyde immediately before loading onto a nylon membrane through a vacuum manifold. IMPORTANT:Protocol 10: Mapping RNA with Nuclease S1 Preparations of RNA containing an mRNA of

12、 interest are hybridized to a complementary single-stranded DNA probe. At the end of the reaction, nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by either autoradiography or Southern hybrid

13、ization. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5 and 3 ends of mRNAs on cloned DNA templates. IMPORTANT:Protocol 11: Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes Preparations of RNA containin

14、g an mRNA of interest are hybridized to a radiolabeled single-stranded RNA probe. At the end of the reaction, a mixture of RNase A and RNase T1 is used to degrade unhybridized regions of the probe, and the surviving molecules are then separated by denaturing gel electrophoresis and visualized by aut

15、oradiography. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5Protocol 12: Analysis of RNA by Primer Extension Primer extension is used chiefly to map the 5 termini of mRNAs. A preparation of polyadenylated mRNA is first hybridized with a

16、n excess of a single-stranded oligodeoxynucleotide primer, which is complementary to the target RNA and radiolabeled at its 5 terminus. Reverse transcriptase is then used to extend the 3 end of the primer. The size of the resulting cDNA, measured by denaturing polyacrylamide gel electrophoresis, is

17、equal to the distance between the 5 end of the priming oligonucleotide and the 5 terminus of the target mRNA.Chapter 7, Protocol 1Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction Prepare all reagents used in this protocol with DEPC-treated H2O.

18、CAUTIONRECIPEMATERIALSBuffers and SolutionsChloroform:isoamyl alcohol （49:1, v/v） Ethanol Formamide （Optional） Deionized formamide is used for the storage of RNA.Isopropanol Liquid nitrogen Phenol PBS Required for cells grown in suspension and monolayers only.Sodium acetate （2 M, pH 4.0） Solution D

19、（denaturing solution） Cells and TissuesMammalian cells Mammalian tissue samples METHOD 1. Prepare cells or tissue samples for isolation of RNA as appropriate for the material under study. The table below describes the amounts of Solution D required for each type of sample.Amount of Solution D Requir

20、ed to Extract RNA from Cells and Tissues Amount of Tissueor CellsAmount ofSolution D100 mg of tissue3 mlT-75 flask of cells60-mm plate of cells1 ml90-mm plate of cells2 ml2. For tissues a.Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.b.Transfer approx. 100 m

21、g of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tissue using a pestle. The tissue can be kept frozen during pulverization by the addition of liquid nitrogen.c.Transfer the powdered tissue to a polypropylene snap-cap tube containing 3 ml of Solution D.d.Homogenize the

22、tissue for 15-30 seconds at room temperature with a polytron homogenizer.3. For mammalian cells grown in suspension Harvest the cells by centrifugation at 200-1900g （1000-3000 rpm in a Sorvall RT600 using the H1000 rotor） for 5-10 minutes at room temperature in a benchtop centrifuge.Remove the mediu

23、m by aspiration and resuspend the cell pellets in 1-2 ml of sterile ice-cold PBS.Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 2 ml of Solution D per 106 cells.Homogenize the cells with a polytron homogenizer for 15-30 seconds at room temperature.4. For mammal

24、ian cells grown in monolayers Remove the medium and rinse the cells once with 5-10 ml of sterile ice-cold PBS.Remove PBS and lyse the cells in 2 ml of Solution D per 90-mm culture dish （1 ml per 60 mm dish）.Transfer the cell lysates to a polypropylene snap-cap tube.Homogenize the lysates with a poly

25、tron homogenizer for 15-30 seconds at room temperature.5. Transfer the homogenate to a fresh polypropylene tube and sequentially add 0.1 ml of 2 M sodium acetate （pH 4.0）, 1 ml of phenol, and 0.2 ml of chloroform-isoamyl alcohol per milliliter of Solution D. After addition of each reagent, cap the tube and mix the contents thoroughly by inversion. 6. Vortex the homogenate vigorously for 10 seconds. Incubate the tube for 15 minutes on ice to permit comple