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

320 Nakashima, Akahoshi, and Tanaka
13. 80% ethanol.
14. PEG-NaCl solution (20% PEG6000, 2.5 M NaCl).
15. Microcentrifuge tube (0.5 mL).
16. Microcentrifuge tube (1.5 mL).
17. Equipment and reagents for 4% agarose gel electrophoresis.
18. TBE: 89 mM Tris, 89 mM boric acid, and 2 mM EDTA (pH 8.3).
19. Ethidium bromide (10 µg/mL).
20. Loading buffer: 0.4% bromophenol blue, 0.4% xylene cyanol FF, 50% glycerol in H 2 O.
3. Method
To provide a DNA target for the PCR, cDNA is synthesized using reverse transcriptase.
1. Place 1 µg of RNA in a 0.5-mL microcentrifuge tube, and add 4 µL of 25 mM MgCl 2 ,
2 µL of 10× PCR buffer, 8 µL of 10 mM dNTP, 1 µL of RNase inhibitor, 1 µL of random
hexamers, 1 µL of MuLV reverse transcriptase, and DEPC-treated sterile H 2 O to form
final total volume of 20 µL.
2. Mix and stand for 10 min at room temperature. Add two drops of mineral oil. Briefly
spin down the reaction mixture.
3. Incubate the reaction mixture for 15 min at 42°C, then for 5 min at 99°C, and finally
for 5 min at 4°C.
4. Add 4 µL of 25 mM MgCl 2 , 8 µL of 10× PCR buffer, 65.5 µL of sterile H 2 O, 0.5 µL of
AmpliTaq DNA polymerase, and 1 µL of forward primer (10 µM) and reverse primer
(10 µM ) to the RT product and mix. Briefly spin down the reaction mix.
5. Perform the PCR by subjecting the reaction mixture to an initial denaturation of 3 min
at 94°C, 35 cycles of 1 min at 94°C and 1 min at 60°C, followed by a final extension
of 7 min at 72°C.
6. Discard the mineral oil and transfer the PCR product to a 1.5-mL microcentrifuge tube.
7. Add 60 µL of PEG-NaCl solution to the PCR product. Mix and stand for 10 min at 37°C.
8. Centrifuge at 12,000g for 10 min at 4°C.
9. The DNA precipitate should be visible as a pellet at the bottom of the tube. Remove the
supernatant and wash the pellet with 1 mL of 80% ethanol.
10. Centrifuge at 7500g for 5 min at 4°C.
11. Carefully remove all the supernatant and air dry the pellet.
12. Dissolve the pellet in 100 µL of H 2 O.
3.1. Restriction Enzyme Digestion
1. Mix 5 µL of PCR product, 1 µL of 10× reaction buffer, 1 µL of the appropriate restriction
enzyme (1–10 U), and 3 µL of H 2 O in a 1.5-mL microcentrifuge tube.
2. Incubate the reaction mixture for more than 2 h at the optimum temperature for enzyme
activity.
3.2. Analysis of RFLP
1. Prepare an appropriate concentration of agarose gel containing 0.2 µg/mL of ethidium
bromide for electrophoresis with TBE buffer.
2. Load the gel with 10 µL of digested PCR product mixed with 1 µL of 10× loading buffer,
5 µL of undigested PCR product mixed with 4 µL of H 2 O, and 1 µL of 10× loading buffer
and a DNA size standard.
3. Perform electrophoresis and inspect the gel under an ultraviolet transilluminator (Fig. 1).