John M. S. Bartlett.pdf - Bio-Nica.info

206 Bartlett
Table 1
Example Assay Layout
Sample
Control 10 pg of RIα 100 pg of RIα Sample 1 Sample 2 Sample 3 Etc.
0.1 pg of RIα
mutant
1.0 pg of RIα
mutant
10 pg of RIα
mutant
100 pg of RIα
mutant
8. Human placental ribonuclease inhibitor (40 U/µL, Pharmacia, UK).
9. Radioactive dNTP mix: 1.25 mM each of dATP, dTTP, dGTP, and 0.5 mM dCTP in distilled
water, aliquoted, and stored at –20°C (Life Technologies, Paisley, UK). Before use, add
0.1 µCi 32 P dCTP (Amersham, UK)/5 µL dNTP mix to be used in PCR (see Note 5).
10. Taq polymerase (5 units/µL) and buffer (Applied Biosystems, UK) 10× buffer: 500 mM
potassium chloride, 100 mM Tris-HCl, 1% Triton-X and 25 mM magnesium chloride
(see Note 6).
11. Paraffin oil (see Note 7).
12. Sodium chloride (100 mM) in sterile distilled water.
13. EcoRV restriction enzyme 10 units/µL (Pharmacia UK).
14. PAGE electrophoresis system (e.g., Protean II, Bio-Rad UK).
15. Gel fixative: 5% glacial acetic acid, 40% methanol, 10% glycerol in water.
16. Gel dryer.
3. Methods
3.1. Reverse Transcription
1. Add 1 µL of random hexamer (100 ng) to 1 µg of RNA and make up to a total volume of
9.5 µL with distilled water in a 0.2-mL thin-walled PCR tube.
2. Heat to 65°C for 10 min and cool on ice.
3. Prepare reverse transcription mix as follows: 4 µL of 5× reverse transcriptase buffer, 5 µL
of 2 mM dNTP mix, 1 µL of reverse transcriptase (200 units), 0.5 µL of human placental
ribonuclease inhibitor per reverse transcription reaction.
4. Incubate at 42°C for 1 h, inactivate reverse transcriptase at 80°C for 5 min, and store
cDNA at –20°C until required for quantitative PCR (see Note 8).
3.2. Quantitative PCR
1. Construction of standard reactions: Duplicate reactions were set up as follows: to separate
aliquots of 10 pg of control RI alpha (in 10 µL), add 0.1, 1, 10, and 100 pg of mutant RI
alpha (in 10 µL). Also, to separate aliquots of 100 pg of control RI alpha (in 10 µL), add
0.1, 1, 10, and 100 pg of mutant RI alpha (in 10 µL). Prepare separate tubes only containing
10 pg of mutant plasmid or 10 pg of control plasmid (restriction digest controls).
2. For each sample (unknown concentration), take 1 µL of cDNA from the reverse transcription
reaction plus 9 µL distilled water and to separate aliquots add 0.1, 1, 10, and 100 pg of
mutant RI alpha (in 10 µL; see Table 1 for assay layout; also, see Note 9).