3.7. To the first strand mix add the following:
15.00 μl 10 x cDNAII buffer
3.50 μl DNA Polymerase I (10U/μl) (Invitrogen)
1.50 μl RNase H (2U/μl) (Invitrogen)
1.00 μl dNTPs (25 mM)
98.00 μl H2O
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150.00 μl Incubate for 2 h at 16℃.
3.8. Use a 5μl aliquot of this sample and check on an agarose gel: a clear DNA-smear between 500-4000 bp should be visible (see figure). Phenol-Chloroform extract the rest of the cDNA sample (145μl) and precipitate with 0.6 vol. isopropanol.
3.9. Resuspend the cDNA in 40μl H2O. Half this amount can then be used for template preparation and half stored for future use.
Single and double stranded cDNA;
4. Template preparation
4.3. Mix the following restriction digests:
20.00 μl cDNA
1.00 μl Enzyme (1) – TaqI (10U)
8.00 μl R/L buffer (5 x)
11.00 μl H2O
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40.00 μl
Incubate at the temperature appropriate for the Enzyme (1) for 1 h. (65℃).
