Synthesis Of Doublestranded cDNAs From Subtracted cDNAs

1. Set up the following reaction: Subtracted first-strand cDNAs, 1 mg Second-strand 10X buffer, 50 ml Poly(A)j2 t0 20 primers, 0.2 mg Escherichia coli DNA polymerase I, 20 units Nuclease-free dd.H2O to a final volume of 50 ml

2. Set up a tracer reaction for the second-strand DNA by transferring 5 ml of the mixture to a fresh tube containing 1 ml 4 mCi [a-32PJdCTP (>400 Ci/mmol). The tracer reaction will be used for TCA assay and alkaline agarose gel electrophoresis to monitor the quantity and quality of the synthesis of the second-strand DNA, as described previously.

3. Incubate both reactions at 14°C for 3.5 h and then add 95 ml of 50 mM EDTA to the 5-ml tracer reaction.

4. Heat the unlabeled (nontracer portion) double-strand DNA sample at 70°C for 10 min to stop the reaction. At this point, the double-strand DNAs should be produced. Briefly spin down to collect the contents at the bottom of the tube and place on ice until use.

5. Add 4 units of T4 DNA polymerase (2 u/mg input cDNA) to the mixture and incubate at 37°C for 10 min. This step functions to make the blunt-end double-stranded cDNAs by T4 polymerase. The blunt ends are required for later adapter ligations.

6. Stop the T4 polymerase reaction by adding 3 ml of 0.2 M EDTA and place on ice.

7. Extract the cDNAs with one volume of TE-saturated phenol/chloroform. Mix well and centrifuge at 11,000 x g for 4 min at room temperature.

8. Carefully transfer the top aqueous phase to a fresh tube and add 0.5 volume of 7.5 M ammonium acetate or 0.15 volume of 3 M sodium acetate (pH 5.2). Mix well and add 2.5 volumes of chilled (-20°C) 100% ethanol. Gently mix and precipitate the cDNAs at -20°C for 2 h.

9. Centrifuge at 12,000 x g for 5 min and carefully remove the supernatant. Briefly rinse the pellet with 1 ml of cold 70% ethanol, centrifuge at 12,000 x g for 5 min and aspirate the ethanol.

10. Dry the cDNA pellet under vacuum for 15 min. Dissolve it in 20 ml of TE buffer. Measure the concentration of the cDNAs as described previously. Store the sample at -20°C until use. Note: Prior to adapter ligation, it is strongly recommended that the quantity and quality of the double-stranded (ds) cDNAs should be checked by TCA precipitation and gel electrophoresis as described previously.

Materials Needed

3 M Sodium acetate buffer, pH 5.2 Ethanol (100% and 70%) Chloroform:isoamyl alcohol (24:1) 0.2M EDTA

Trichloroacetic acid (5% and 7%)

Hybridization Buffer (2X) 40 mM Tris-HCl, pH 7.7 1.2 M NaCl

1 mg/ml Carrier yeast tRNA (optional)

Phosphate Buffer (Stock)

0.5 M Monobasic sodium phosphate

Adjust the pH to 6.8 with 0.5 M dibasic sodium phosphate.

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