In a 200ul PCR epp.-tube add:
 

• x ul RNA (1ug RNA total)
• 1 ul (dATP 10mM stock)
• 1 ul (dTTP 10mM stock) or 3 ul of a 30mM stock with dATP, TTP, & GTP
• 1 ul (dGTP 10mM Stock)
• 4 ul GenoSys cDNA primers for E. coli
• (GenoSys primers must be resuspended in 40 ul RNase free H₂O prior to use. Separate the 40 ul of cDNA primers into 4 ul aliquots, storing aliquots in 200 ul PCR tubes at 4^oC until ready for use.)
• 6 ul 5x First strand buffer
• x ul RNase free H₂O 
• 23 ul total

1. Anneal cDNA to RNA template by placing above reaction into ThermoCycler using program named Array.

90^oC for 2 min

42^oC for 20min

2. After 20 min at 42^oC, add to the above reaction:

2 ul -P³³ dCTP (2000-3000 Ci/mmol) NEN cat# NEG-613H (20 uCi)

3 ul 0.1M DTT

1 ul Superscript II RNase H^- Reverse Transcriptase 200 units/ul (BRL cat# 18064-022)

1 ul Ribonuclease Inhibitor, Cloned 10 units/ul (BRL cat# 15518-012)

30 ul Total volume

3. Mix by pipetting and place in radioactive heat block at 42^oC for 2-3hrs.

4. After incubation, denature labeling reaction by adding to the reaction:

2 ul 0.5 M EDTA

3 ul 5 N NaOH

Incubate at 65^oC for 30 min

5. Neutralize with:

8 ul 1 M Tris (pH 7.4)

7 ul 2 N HCL

6. Take out 1.0 ul, dilute 1:10 in TE, and determine total cpm from the 1:10 dilution.

7. Remove the unincorporated-labeled nucleotides by passing the reaction over a Sephadex G-50 gravity column.

a. Fill small glass pasture pipette to meniscus pinch with TE saturated G-50.

b. Wash column 4-5X with 400 ul of 10mM TE buffer pH 8.0.

c. Add the 30 ul labeled reaction to the column and elute with 10mM TE buffer at 200 ul intervals. Collect the 200 ul fractions in individual 1.5 ml epp-tubes. Pool the first 2-3 hot fractions (indicated by a Geiger counter), and use this for the hybridization probe (pool 3 fractions if the first fraction is only slightly hot - pool this fraction and the next 2 fractions).
 

Determine the % of radioactive incorporation

% incorporation= unlabeled cpm/ul x total volume x 100

Total cpm x 10 (dil.) x 50 (vol. of labeling rxn)
 

G-50 Solution TE solution

5g G-50 10mM Tris (pH 8.0)

75 mls TE 1mM EDTA
 

2N HCl

1.67ml of 12N HCl stock

8.33ml dH₂O