Mix well and dispense in 100 ul aliquots and store at -70 C.
2.Incubate the reaction at 40 C for 1.0 hour.
3.Terminate reaction by placing on ice and extract with 100 ul of phenol- chloroform. Remove 95 ul of aqueous and reextract organic phase with another 100 ul of O.1 X sterile TE
4.Precipitate the DNA:RNA hybrid with 0.1 vol sodium acetate (20 ul) and two volumes (440 ul) of ethanol. Chill in an ice bucket for 10 min. and pellet by centrifugation for 20 minutes in the cold.
5.Drain ethanol and resuspend pellet in 20 ul of TE and precipitate with ammonium acetate (20 ul of ammonium acetate, 80 ul of ethanol). Allow to stand at least 2-3 hrs (O/N is better according to BRL). Spin down at room temperature. Wash pellet in 70% ethanol.
6. Dry in the Speed Vac and resuspend in 10 ul of O.lX TE .
(c)SECOND STRAND SYNTHESIS
(Polites and Marrotti, Biotechniques 1986, 4:514-520)1. To the dried cDNA pellet add the following:
1st strand cDNA 10.0 ul 0.4M Hepes pH 7.6 25.0 ul 6O mM MgCl2 10.0 ul 1.0 M DTT 1.0 ul 1.O M KCl 6.0 ul 5.O mM dNTP mix 10.0 ul 10 mM B-NAD 1.5 ul P32-dCTP 10.0 ul DNA Pol 1 15.0 ul RNase H 3.0 ul E. coli ligase 1.0 ul dH20 7.5 ul
2. Mix well on ice and incubate at 14 C for 1.0 hr. then shift to RT for an additional hour.
3. Stop the reaction by placing on ice. Perform one phenol/chloroform extraction (reextracting the organic layer) and precipitate once with sodium acetate and ethanol. Dry the pellet and resuspend in 5 ul of O.1X TE.
(d)BLUNT ENDING THE cDNA
1. To the cDNA add the following:1OX NTB (Maniatis nick-translation buffer) 3 ul 5mM dNTPs 1 ul H20 20 ul T4 DNA polymerase 0.5 ul2.Incubate at 37 C for 20 min. Adjust aqueous volume to 100 ul (by adding 70 ul of O.1 X TE). Perform one phenol chloroform extraction, reextracting the aqueous volume and precipitate the DNA by ammonium acetate precipitation. Wash pellet with 700/0 ethanol and dry in speed vac. Resuspend cDNA in 10 ul of O.1 X TE.
(e)ADDING LINKERS
1. We prepare and test linkers as in Sambrook et al. (1989).
Combine the following on ice.
double -stranded cDNA 10.0 ul 1O X Ligation buffer 2.0 ul Kinased linkers 5.0 ul H20 2.0 ul 1OmM ATP 0.5 ul T4-DNA ligase 0.5 ul
Mix well and incubate at 140C overnight. Check all centrifuges and work area for radioactivity.
(f.)SIZE FRACTIONATION OF cDNA AND LIGATION TO LAMBDA
1. Save 0.5 ul of the ligation mix in 5 ul of TE with bromphenol blue stop dye.2. Heat inactivate ligase by heating at 65 C for 10 min.
3. To the ligation mix add:
1O X Restriction buffer 10.0 ul 1OX Gelatin (1 mg/ ml) 10.0 ul H2O 56.0 ul Xbal 2.0 ul
Incubate for 1.0 hr at 37 C
Add 2.0 ul of EcoRl and continue incubation for 2 more hours. During this time set-up a 5% acrylamide gel. (Note: This step for directional Eco-Xba cDNA cloning)
3. Remove restriction digest to ice and store at -20 C. Remove 3.0 ul for gel analysis. Combine with 5 ul of TE with bromphenol blue stop dye.
