cDNA Synthesis with M-MLV RT 87
- Add 10 I.tL of 0.5M EDTA to the remaining reaction mixture on ice.
The product can now be phenol-extracted and ethanol-precipitated in
preparation for addition of linkers or adapters (35,37,49).
4.3.3. One-Tube Double-Stranded eDNA Synthesis:
First-Strand Reaction in Preparation
for Amplification of Specific eDNA Sequences by PCR - The following protocol is designed for synthesis of first-strand eDNA suit-
able for PCR amplification with specific primers. For strategies designed
for PCR amplification of total eDNA libraries, see ref. 50 and Chapter 4. - In a 0.5-mL tube, mix on ice 1 lag total RNA (smaller amounts of poly
[A]+ mRNA can be used) and one of the following: 0.5 lag pd(T)12_lS,
or 100 ng pd(N) 6. Add water to a vol of 14 pL; heat at 70°C for 10 min
and chill on ice. - In the tube on ice, mix 2 I.tL 10X DNA Synthesis Buffer, 2 laL 0.1M
DTT, and 1 laL 10 mM dNTPs. - If pd(T)12_18 is used, equilibrate the tube to 37°C, and add 200 U (1 ~L) of
SuperScript TM RT; ifpd(N)6 is used, immediately add 1 ]xL of SuperScripff M
RT, and incubate at room temperature for 10 min; finally, incubate all
reaction types at 37°C for 1 h. - The final reaction composition is 20 mM Tris-HC1 (pH 8.3), 50 mM
KC1, 2.5 mM MgC12, 10 mM DTT, 500 ~/each of dATP, dCTP, dGTP,
and dTTP, 50 lag/mL RNA, 25 ~tg/mL pd(T)12_18 or 5 lag/mL pd(N) 6,
100 I.tg/mL BSA, and 10,000 U/mL SuperScript TM RT. - Heat the tube at 90°C for 5 min, place the tube on ice for 5 min, and
collect the tube contents by brief centrifugation. - Add 2 ~L of RNase H (2 U/laL) to the tube, and incubate for 20 min at
37°C before proceeding with PCR amplification. Digestion of the first-
strand cDNA-RNA hybrid to remove RNA is essential to subsequent
amplification of the cDNA. - Place the tube on ice. Add directly to the tube, and mix 8 laL 10X DNA
Synthesis Buffer, appropriate amounts of PCR primers (51,52), suffi-
cient water to bring the final vol to 100 IlL after Taq DNA polymerase
is added, and Taq DNA polymerase (1-4 U). - Layer 100 IlL of mineral oil over the reaction mixture.
- Carry out PCR amplification (51,52).
4.3.4. [32p]cDNA Synthesis- The following protocol is designed to synthesize high specific activity
(4 x 108 cprrdlag) [32p]cDNA that represents all sequences in an RNA
population. The ratio of random primer to RNA template has been se-