1. Ethidium bromide is a suspected mutagen, and should be handled and disposed accordingly.
2. The preparation of Phenol-SEVAG is described in detail in ref. 15. For convenience, this reagent can also be purchased ready-to-use from USB, 0.1% 8-hydroxiquinolin (w/v) can be added to prevent oxidation of phenol and for easy visualization of the organic phase.
3. Owing to the large amounts of reverse transcriptase used, cloned MuLTV RT, is preferred over AMV RT because the former is considerably cheaper, although considered of lower performance when extending long products (i.e., when synthesizing cDNA to construct a library).
4. Purification of synthetic oligonucleotides: If the oligonucleotide has the trityl group on, detritylate it by resuspending the dry pellet in 200 |L 80% acetic acid. Leave at room temperature for 30 min. Dry in a Speed-Vac. Resuspend with 50 |L dH2O, 50 |L, deionized formamide, and 25 |L of formamide loading dye. For a 15- to 25-mer, prepare a 2-mm thick 15% sequencing acrylamide, 1X TBE gel in a protein gel cast. Use a wide well (5 cm), or use several smaller wells. Heat the sample at 100°C for 2 min and load. Run at 250 V until bromophenol blue (the fastest migrating dye) is at 2 cm of the bottom (if in doubt, check ref. 15, for the relative position of fragments in denaturing acrylamide gels according to the migration of the dyes). Disassemble the cast, place the gel on Saran Wrap and on a silica gel TLC plate with fluorescence indicator (Sigma, T-6270), and illuminate the gel with a handheld UV lamp. Wear appropiate eye protection when using short wave UV light. The DNA will appear as a shade, since it absorbs the UV light that stimulates fluorescence on the TLC plate. Degradation and unfinished synthesis products will also appear, but the bona fide band should represent over 60% of the total. Cut the gel containing the DNA with a scalpel, and mince the gel piece thoroughly. Place the puree in a 15-mL conical tube, and add 2 mL of dH2O and 1 mL of TE. Shake the tube overnight at 37°C. On the next day, recover the liquid by centrifugation, and concentrate with n-butanol. This is done by adding equal volumes of n-butanol to the DNA solution, mixing well, and spinning in a tabletop centrifuge. Discard the top butanol layer, and repeat until the final volume is 1 mL. Make sure now that you are using a polypropylene tube. Add 5 mL of chloroform, mix, spin, and recover the top layer. Adjust the volume to 1 mL, and apply the sample to a Sephadex G25 NAP-10 column (Pharmacia) previously equilibrated with H2O. Elute the sample with 1.5 mL of dH2O, and dry in the Speed-Vac. Resuspend the pellet in 200 |L of dH2O, and measure the absorbance of 2 ||L in 1 mL of H2O at 260 nm (1 OD260 = 30 |g/mL oligo). Calculate the concentration in mol/mL (the average molecular weight of a nucleotide is 330 g/mol).
5. Special care should be taken when handling radioisotopes. Refer to a laboratory safety manual for proper handling and disposal of 32P.
6. Never autoclave solutions containing ammonia. This solution should be filter-sterilized through a 0.2-| filter.
7. (See also Subheading 3.9. and Chaper 37) This is also available as a commercial kit. Make sure the primers contained in the kit are random hexamers (i.e., Boehringer), since longer primers will label short DNA fragments less efficiently.
8. After preparation, sonicate extensively to shear the DNA and store at -20°C. DNA can also be sheared by passing multiple times through a 21-gage needle until the material flows easily through the needle.
9. This kit includes vector, T4 DNA ligase, 10X ligation buffer, and competent cells in order to clone the PCR clones obtained. Although expensive, its use greatly facilitates the cloning of PCR products.
10. RNA can be rapidly degraded by contaminating ribonucleases. For this reason, RNase-free material must be used, and gloves should be worn throughout the whole procedure.
12. If oligodT was used for cDNA synthesis, then add the arbitrary primer at this point. The optimal primer concentration in the PCR reaction ranges from 0.3-10 |M. This parameter will depend on the primer sequence and on the particular primer preparation used. To minimize effects of the latter, purification of oligonucle-otides through denaturing acrylamide gels is recommended. In a typical reaction, we use the primer at 1 ||M.
13. This precipitation step is added to remove most of the unincorporated nucleotide that would contaminate the lower buffer during electrophoresis. It also concentrates the product so that autoradiography can be performed in a shorter period of time.
14. Preparation of a sequence marker. Digest pBR322 with MspI. After digestion, heat-inactivate the enzyme at 65°C for 20 min. Remove an aliquot containing approx 100 ng and label in 20 ||L containing: 2 |L 10X First strand buffer, 2 |L 0.1 M DTT, 3 |L of a32P dCTP, and 1 |L of MuTLV RT. Bring the volume to 20 |L with dH2O. Incubate at 37°C for 1 h. Add 1 |L of 0.2 M EDTA, 1 |L 10 mg/mL oyster glycogen, 22 |L of 5 M ammonium acetate, and 132 |L of 100% ethanol. Precipitate 5 min at room temperature, spin for 15 min at 12,000 rpm, carefully discard the supernatant, and resuspend the pellet in 50 | L of formamide loading dye. Place 1 mL in a tube and count it without scintilation fluid in a scintillation counter. Load 3000-5000 cpm/gel. The bands generated are (in bp): 622, 527,
404, 307, 242, 238, 217, 201, 190, 180, 160 + 160, 147 + 147, 123, 110, 90, 76, and 67. Some double bands may appear if the marker has not been well denatured. This marker will be stable for a long time at -20°C, but the half-life of the isotope has to be considered (14.3 d).
15. Instructions for the preparation and casting of this type of gels are provided in ref. 15.
16. Several bands may appear if the original PCR product is not homogeneous. Also, a single gene may give rise to several mRNAs.
17. The size of ribosomal RNAs varies among species.
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