RNA Target Labeling
October 09, 2000
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 4oC 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.
90oC for 2 min
42oC for 20min
2. After 20 min at 42oC, 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 42oC
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 65oC 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
|